Recombinant adenoviral vectors expressing chimeric fiber proteins for cell specific infection and genome integration

ABSTRACT

The present invention provides for novel chimeric Ad-vectors carrying transgene, or portions of transgenes for stable and efficient gene transfer into diverse cell types or tissues in a CAR- and/or α υ β 3/5 -independent manner. Also provided are methods for producing such vectors and the use thereof for gene therapy to target a specific cell type or tissue.

This application is an application filed under 35 U.S.C. §371 which isbased on International Application No. PCT/US00/15442, filed Jun. 1,2000, which claims the priorities of provisional applications U.S. Ser.No. 60/137,213, filed Jun. 1, 1999 and U.S. Ser. No. 60/161,097, filedOct. 22, 1999, the contents of all of which are hereby incorporated byreference in their entirety into the present application.

This invention was made, at least in part, with funding from theNational Institutes of Health (Grant Nos. R01 CA 80192-01 and R21 DK55590-01). Accordingly, the United States Government has certain rightsin this invention.

FIELD OF THE INVENTION

This invention relates to the field of gene therapy, and in particular,to novel adenovirus (Ad) vectors that selectively infect cells for genetherapy, and to Ad vectors containing modifications of the fiber proteinto allow retargeting of any adenovirus serotype.

BACKGROUND OF THE INVENTION

Gene transfer vectors require the efficient transduction of targetcells, stable association with the host genome, and adequate transgeneexpression in the appropriate target cell, without associated toxic orimmunological side effects. Currently available viral vector systems,including recombinant retroviruses, adenoviruses and adeno-associatedviruses, are not suitable for efficient gene transfer into many celltypes. Retroviral vectors require cell division for stable integration.Recombinant adenoviruses are not able to infect many cell typesimportant for gene therapy, including hematopoietic stem cells,monocytes, T- and B-lymphocytes. Moreover, recombinant adeno-associatedvectors (AAV) integrate with a low frequency.

First generation adenoviruses have a number of properties that make theman attractive vehicle for gene transfer (Hitt, M. M. et al. 1997Advances in Pharmacology 40:137–205). These include the ability toproduce purified virus at high titers in concert with highly efficientgene transfer of up to 8 kb long expression cassettes into a largevariety of cell types in vivo, including non-dividing cells. Limitationsof first generation adenoviruses include the development of immuneresponses to expressed viral proteins resulting in toxicity and virusclearance. The episomal status of adenoviral DNA within transduced cellsis another limitation of first generation Ad vectors. Stable integrationof adenovirus DNA into the host genome is reported only for wild-typeforms of specific subtypes and appears not to occur in a detectablemanner with E1/E3-deleted Ad 5 (adenovirus serotype 5) vectors widelyused for gene transfer in vitro and in vivo [Hitt, M. M. et al. 1997Advances in Pharmacology 40:137–205].

Recombinant AAV vectors (rAAV) integrate with a low frequency (about 1out of 20,000 genomes) randomly as cocatemers into the host genome(Rutledge, E. A.; Russel, D. W. 1997 J. Virology, 71, 8429–8436). Thepresence of two AAV inverted terminal repeats (ITRs) and as yet unknownhost cellular factors seem to be the only requirement for vectorintegration (Xiao, X., et al, 1997, J. Virology, 71, 941–948; Balague,C., et al. 1997, J. Virology, 71, 3299–3306; Yang, C. C. 1997, J.Virology, 71, 9231–9247). In the presence of the large AAV Rep proteins,AAV integrates preferentially into a specific site at human chromosome19, called AAVS1 (Berns, K. I., 1996, Fields Virology, Fields, B. N. etal. (ed) Vol. 2, Lippincott-Raven, Philadelphia, Pa., 2173–2220). TheAAV capsid is formed by three coat proteins (VP1–3), which interact withspecific heparin sulfates on the cell surface and probably with specificreceptor(s). However, many cell types, including hematopoietic stemcells, lack these structures so that rAAV vectors based on AAV2 cannotinfect or transduce these cells (Malik P. et al., 1997, J. Virology, 71,1776–1783; Quing, K. Y., et al. 1998, J. Virology, 72, 1593–1599). Otherdisadvantages of rAAV vectors include the limited insert size (4.5–5 kb)that can be accommodated in rAAV vectors lacking all viral genes and lowtransducing titers of rAAV preparations.

Adenovirus infection is initiated by attaching to the cell surface of Ad5 via its fiber protein (for a review, see Shenk, T. 1996 FieldsVirology, Vol. 2, Fields, B. N. et al. (ed) Vol. 2, Lippincott-Raven,Philadelphia, Pa., 2111–2148). The distal, C-terminal domain of thetrimeric fiber molecule terminates in a knob, which binds to a specificcellular receptor identified recently as the coxackie-adenovirusreceptor (CAR) (Bergelson, J. M. et al. Science, 275, 1320–1323). Afterbinding, in an event independent of virus attachment, Arg-Gly-Asp (RGD)motifs in the penton base interact with cellular integrins of the α3 andβ5 types. This interaction triggers cellular internalization whereby thevirion achieves localization within the endosome. The endosomal membraneis lysed in a process mediated by the penton base, releasing thecontents of the endosome to the cytoplasm. During these processes, thevirion is gradually uncoated and the adenoviral DNA is transported tothe nucleus where replication takes place. The terminal protein, whichis covalently attached to the viral genome and the core protein V thatis localized on the surface of the cores have nuclear localizationsignals (NLSs) (van der Vliet, B. 1995, The Molecular Repertoir ofAdenoviruses, Vol. 2, Doerfler, W. and Boehm, P. (ed.), Springer Verlag,Berlin, 1–31). These NLSs play a crucial role in directing theadenoviral genome to the nucleus and probably represent the structuralelements which allow adenovirus to transduce non-dividing cells. Whenthe double-stranded, linear DNA reaches the nucleus, it binds to thenuclear matrix through its terminal protein.

Since the cell types that can be infected with Ad5 or Ad2 vectors arerestricted by the presence of CAR and specific integrins, attempts havebeen made to widen the tropism of Ad vectors. Genetic modification ofadenovirus coat proteins to target novel cell surface receptors havebeen reported for the fiber (Krasnykh, V. et al. 1998 J. Virology, 72,1844–1852, Krasnykh, V. et al. 1996 J. Virology, 70, 6839–6846,Stevenson, S. D., et al. 1997, J. Virology, 71, 4782–4790), penton base(Wickham, T. J., et al. 1996, J. Virology, 70, 6831–6838; Wickham, T.J., et al. 1995, Gene Therapy, 69, 750–756), and hexon proteins(Crompton, J., et al. 1994, J. Gen. Virol. 75, 133–139). The mostpromising modification seems to be the functional modification of thefiber protein or more specifically of the fiber knob as the moiety,which mediates the primary attachment. Two groups have reported thegeneration of fibers consisting of the Ad5 tail/shaft and the knobdomain of Ad3 (Krasnykh, V. et al. 1996 supra, Stevenson, S. D., et al.1997, supra). Recently, recombinant adenoviruses with fibers containingC-terminal poly-lysine, gastrin-releasing peptide, somatostatin,E-selectin-binding peptide, or oligo-Histidines were produced in orderto change the native tropism of Ad5. Krasnikh et al. found (Krasnykh, V.et al. 1998 supra) that heterologous peptide ligands could be insertedinto the H1 loop of the fiber knob domain without affecting thebiological function of the fiber. Based on studies with other Adserotypes, it appears that the length of the fiber shaft is a criticalelement, determining the efficiency of interaction with cell surfaceintegrins and the internalization process. Thus far, there is noreported data demonstrating successful retargeting of Ad5 vectors for aspecific cell type.

Therefore, there is a present need for an improved adenovirus vectorwhich can be targeted efficiently to a variety of cell types and tissuesand remain stably integrated in the host genome with minimalantigenicity to the host. The present invention discloses novel chimericadenoviral (Ad) Ad-AAV vectors, which express a modified fiber proteinon their capsid, for specifically targeting the vector. Methods ofmaking, uses and advantages of these vectors are described. In addition,the alteration described for the knob and shaft domains of the fiberprotein provide a novel approach to retarget any adenovirus serotype forcell specific infection.

SUMMARY OF THE INVENTION

The present invention provides for novel chimeric Ad-vectors carryingtransgene, or portions of transgenes for stable and efficient genetransfer into diverse cell types or tissues in a CAR- and/orα_(υ)β_(3/5)-independent manner. Also provided are methods for producingsuch vectors and the use thereof for gene therapy to target a specificcell type or tissue.

The recombinant adenovirus vectors of the invention (Example I) providea novel design that allows for the easy production and delivery of a“gutless” adenoviral vector with the added advantage of stableintegration of the transgene into the host genome of different celltype. The adenoviral vector described is devoid of all adenoviralsequences except for the 5′ and 3′ cis elements necessary forreplication and virion encapsidation. The adenovirus-associated virussequences of the invention comprising the 5′ (right) and 3′ (left)inverted terminal repeats (ITRs) flank the transgene gene cassette suchthat they direct homologous recombination during viral replication andviral integration into the host genome. In one embodiment AAV-ITRflanking sequences are used. The vector also contains a selectedtransgene(s) operably linked to a selected regulatory element and apolydenylation stop signal, which is in turn flanked by the flankingsequences described above. The selected transgene(s) can be linked underthe same regulatory elements or under separate regulatory elements inthe same orientation or in opposite orientations with respect to eachother. The selected transgene(s) are any gene or genes which areexpressed in a host cell or tissue for therapeutic, reporter orselection purposes. This vector is characterized by high titer transgenedelivery to a host cell and the ability to stably integrate thetransgene into the host genome. Also provided is a method to improve theintegration frequency and site specific integration by incorporating anAAV rep protein into the recombinant hybrid vector.

The invention also provides chimeric fiber proteins (Example II), whichincludes naturally occurring fiber proteins in which a portion orportions of the sequence are modified to alter cell or tissuespecificity of infection. Altered fiber protein sequences can includefiber protein domains (the knob domain, the shaft domain, and the taildomain) from other or the same adenovirus serotypes or from randomlyselected peptides. A chimeric fiber protein can be entirely composed ofnon-naturally occurring sequences. The invention further relates tonucleic acid sequences encoding the chimeric fiber proteins. Thesenucleic acid sequences can be naturally occurring, a mixture ofnaturally occurring and non-naturally occurring sequences, or entirelynon-naturally occurring sequences.

The heterologous fiber protein sequences described herein can beinserted into any adenovirus based vector which contains a capsid,rendering the virus capable of specifically infecting a given cell ortissue. Adenoviral vectors having such a heterologous fiber sequence canbe used to direct gene transfer into desired cells. For stableintegration of the transgene cassette into the host gemone, the chimericAd-AAV vector described in the invention is the preferred vector of use.

The invention also includes a library of adenoviruses displaying randompeptides in their fiber knobs can be used as ligands to screen for anadenovirus variant with tropism to a particular cell type in vitro andin vivo.

The chimeric Ad-vectors described herein include the Ad.AAV genome witha modified fiber protein expressed on its capsid. These chimeric vectorsare designed to infect a wide variety of cells, in particular, the cellswhich can only be poorly transduced by the commonly used retroviral, AAVand adenoviral vectors. These cells include, but not limited to,hematopoietic stem cells, lung epithelial cells, dendritic cells,lymphoblastoid cells, and endothelial cells. Hematopoietic stem cellssuch as CD34+ cells can be targeted for gene therapy of sickle cellanemia and thalasemia using the vector described herein. The chimericAd-AAV vector capable of transducing genes into endothelial cells can beused in gene therapy for vascular diseases such as atherosclerosis orrestinosis after coronary artery surgery.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A–1C display a proposed mechanism for forming of ΔAd.AAV1 genome.

FIGS. 2A and 2B show electron photomicrographs of hybrid virusparticles: FIG. 2A shows Ad.AAV1 and FIG. 2B shows ΔAd.AAV1.

FIG. 3 illustrates analysis of ΔAd.AAV1 genomes after transduction ofSKHep1 cells. Pulse field gel eletrophoresis (PFGE). 1×10⁶ control SkHep1 cells (SKHep1) (lanes 1–3, 5, 9). SKHep1 cells from G418 resistantpools (ΔAd.AAV1) (infected with ΔAd.AAV1 and selected for 4 weeks)(lanes 6–8, 10–12), or SKHep1 cells collected at 3 days after infectionwith 2000 genomes Ad.AAV1 (Ad) lanes 4, 13) are sealed in agaroseplaques, lysed in situ and subjected to PFGE with or without priordigestion with restriction endonucleases. Southern Blot is performedwith a SEAP specific probe. U=undigested, P=digested with PI-Sce1,I=I-CeuI, E=EcoREI.

FIGS. 4A and 4B show response of K562 and CD34+ cells respectively afterinfection with ΔAd.AAVBG. Cells are incubated for 6 hours with virusunder agitation. At day 3 after infection, transduction frequency iscalculated based on the number of X-Gal positive cells. Viability istested by trypan blue exclusion. N=3, SEM<10%.

FIG. 5 shows Rep expression in SKHep1 and 293 cells after plasmidtransfection. 5×10⁵ cells are transfected with pAAV/Ad, pRSVrep, orpPGKrep by Ca-phosphate co-precipitation. Three days after transfection,cells are harvested. Lysates are separated on a 10% PA gel, followed byWestern Blot with Rep specific antibodies (03-65169), American ResearchProducts), and developed with ECL (Amersham);

FIG. 6 shows detection of vector integration into AAVS1 by PFGE.

FIG. 7 shows strategy for creating an ΔAd.AAV hybrid vector capable ofsite-specific integration. Arrows indicate promoters,(PA)=polyadenylation signal. Ψ=adenoviral packaging signal.

FIGS. 8A–8B shows vectors for transduction studies with SNori asexpression unit and analysis of vector integration on genomic DNA from asmall cell number. Analgous vector sets can be generated withβ-galactosidase (BG) or green fluorescence protein (GFP) as reportergenes.

FIG. 9 shows strategy for substituting the Ad5 fiber sequence by theheterologous fiber X genes using recombination in E. coli.

FIG. 10 shows the expression of CAR and Δ_(v)-integrins on test cells.For flow cytometry analysis, HeLa, CHO, K562, and CD34+ cells wereincubated with monoclonal anti-CAR (RmcB, 1:400 dilution) oranti-Δ_(v)-integrin antibodies (L230, 1:30 dilution). As a negativecontrol, cells were incubated with an irrelevant mouse monoclonalantibody (anti-BrdU, 1:100 dilution). The binding of primary antibodieswas developed with anti-mouse IgG-FITC labeled conjugates (1:100dilution). Data shown represent the average results of quadruplicateanalyses performed on 10⁴ cells.

FIG. 11 shows the electron microscopy of adenovirus particles. Purifiedparticles from Ad5, 9, and 35 were negative contrast stained andanalyzed at a magnification of 85,000×. Defective particles arehighlighted by arrows.

FIG. 12 shows the analysis of attachment and internalization ofdifferent serotypes to CHO, HeLa, K562, and CD34+ cells. Equal amountsof [³H]-thymidine-labeled virions of Ads 3, 4, 5, 9, 35, and 41(measured by OD₂₆₀, and equivalent to an MOI of 400 pfu per cell forAd5) were incubated for one hour on ice as described in Materials andMethods. Cells were then washed, and the number of labeled virions boundper cell was determined. For internalization studies, viruses were firstallowed to attach to cells for 1 h on ice. Then, unbound viral particleswere washed out. Cells were then incubated at 37° C. for 30 min followedby treatment with trypsin-EDTA and washing to remove uninternalizedviral particles. The data were obtained from two to four independentexperiments performed in triplicate. Note the different scale on theY-axes for CD34+ cells.

FIGS. 13A–13C show attachment and internalization of differentadenovirus serotypes to Hela, CHO and 293 cells respectively.

FIGS. 14A and 14B show attachment and internalization of differentadenovirus serotypes to CD34+ and K-562 cells respectively.

FIGS. 15A–15C shows the analysis of viral replication in K562 and CD34+cells by Southern blot analysis of methylated viral DNA. Replicationstudies were performed with 1×10⁵ K562 cells (A) or CD34+ cells (B),infected with methylated Ad5, Ad9 or Ad35. The lane labeled as “load”represents DNA that was extracted form the media/cell mixtureimmediately after adding the indicated viral dose to cells. Theintensities of bands corresponding to methylated and un-methylated viralDNA indicate that ˜85% of the input virus was methylated. To quantifyadsorption and internalization, DNA analysis was performed after priorincubation of virus with cells at 0° C. (adsorption) or 37° C.(internalization). For dose dependent replication studies, the indicatedviral dose (expressed as the number of genomes) was added to the cells,and cellular genomic DNA together with viral DNA was extracted 16 hoursor 36 hours post-infection for K562 and CD34+ cells, respectively.Identical amounts of sample DNA were analyzed by Southern blot. Forquantification purposes, Ad9 replication was analyzed together with Ad5using an Ad5/9 chimeric probe that hybridizes with DNA of both serotypes(C). The analysis of Ad5 versus Ad35 replication was performed with thecorresponding Ad5/35 chimeric probe. Since separate hybridizations withboth Ad5/35 and Ad5/9 probes gave identical signal intensities for Ad5DNA only one panel is shown for Ad5 replication in test cells. Toproduce distinguishable fragments specific for the methylated ornon-methylated status of viral genomes, Ad5 DNA was digested with Xho I,while Ad9 and Ad35 DNA was digested with Xho I and Hind III. The bandsspecific for methylated (not-replicated) viral DNA were ˜12 kb for Ad9,35 kb for Ad5, and ˜12 kb for Ad35. The fragments specific fornon-methylated DNA were 5.8 kb for Ad9, 6.1 kb for Ad5, and 9.5 kb forAd35. Chimeric Ad5/9 and Ad5/35 DNA fragments (1.8 kb) were used asquantification standards and applied onto gel together with digestedviral/cellular DNA (shown on the left part of the figures).

FIGS. 16A–16B shows the structure of Ad5GFP and chimeric Ad5GFP/F35vectors. A) Schematic diagram of the original E1/E3 deleted Ad5-basedvector with GFP-expression cassette inserted into the E3 region (Ad5GFP)and the chimeric vector Ad5GFP/F35 containing the Ad5/35 fiber gene. The2.2 kb Ad5 fiber gene was replaced by a 0.9 kb chimeric fiber geneencoding for the short shaft and knob of Ad35 by a technique thatinvolved PCR-cloning and recombination in E. coli. Kpn I (K) and HindIII (H) sites localized within or around the fiber genes are indicated.The lower panel shows the detailed structure of the chimeric fiberregion. The Ad5 fiber tail [amino acids (aa): 1–44] were joined in frameto the Ad35 fiber shaft starting from its first two amino acids (GV),which are conserved among many serotypes. A conserved stretch of aminoacids TLWT marks the boundary between the last α-sheet of Ad35 shaft andthe globular knob. The Ad35 fiber chain termination codon is followed bythe Ad5 fiber poly-adenylation signal. The region of Ad5GFP/F35 encodingfor chimeric fiber was completely sequenced with Ad5 specific primers(see Material and Methods). B) Restriction analysis of viral genomes.Viral DNA was isolated from purified Ad5GFP and Ad5GFP/F35 particles asdescribed elsewhere. One microgram of DNA was digested with Hind III orKpn I and separated in ethidium bromide stained agarose gels (leftpanel) which were subsequently blotted and analyzed by Southern blotwith an Ad5 E4 specific probe (nt 32,7775–33,651) (right panel).Specific patterns, designating the correct structure for both viralvectors were detected. The Hind III fragments specific for Ad5GFP andAd5GFP/F35 were 2.9 kb and 4.9 kb, respectively. The Kpn I fragment thatconfirmed the correct Ad5GFP/F35 structure was 1.6 kb compared to a 7.6kb Ad5GFP fragment. M-1 kb ladder (Gibco-BRL, Grand Island, N.Y.).

FIG. 17A–17B shows the generation of ΔAd.AAV genomes by recombinationbetween inverted homology regions. A) Recombination between two invertedrepeats (IRs) present in separate Ad.AAV vectors. The upperfirst-generation Ad.AAV vector (˜34 kb) contains two 1.2 kb IRs flankingGene X. An AAV-ITR (“AAV.ITR”) is located between the Ad packagingsignal (Ψ) and the left IR. The lower Ad.AAV vector, shown in theopposite orientation, contains the same IRs flanking a transgenecassette. An AAV-ITR is located between the left IR and the Ad packagingsignal. During Ad replication, recombination between an IR on eachvector (indicated by an X) mediates the formation of ΔAd.AAV genomes(lower portion of panel A) with the transgene flanked by IRs, AAV-ITRs,Ad packaging signals, and Ad ITRs. These genomes are efficientlypackaged into Ad capsids. The other recombination product (not shown) isa defective Ad.AAV vector lacking packaging signals. B) Recombinationbetween homology regions of Gene X present in separate Ad.AAV vectors.The upper Ad.AAV vector contains a promoter (P) operably linked to the5′ portion of Gene X. An AAV-ITR is inserted between the Ad packagingsignal (T) and the promoter. The lower Ad.AAV vector, shown in theopposite orientation, contains the 3′ portion of Gene X linked to apoly-adenylation region (PA). An AAV-ITR is inserted between the Adpackaging signal (T) and the polyadenylation region. The 5′ portion ofGene X in the upper vector has a region of overlapping homology with the3′ portion of Gene X in the lower vector. Recombination between theoverlapping homology regions (indicated by an X) mediates the formationof ΔAd.AAV genomes with the assembled Gene X flanked by AAV-ITRs, Adpackaging signals, and Ad ITRs.

FIG. 18 shows the structure of Ad5/35. Schematic diagram of the originalE1/E3 deleted Ad50based vector with GFP-expression cassette insertedinto the E3 region (Ad5GFP) and the chimeric vector Ad5GFP/F35containing the Ad5/35 fiber gene. The 2.2 kb Ad5 fiber gene was replacedby a 0.9 kb chimeric fiber gene encoding for the short shaft and knob ofAd35 by a technique that involved PCR-cloning and recombination in E.coli Kpn I (K) and Hind III (H) sites localized within or around thefiber genes are indicated. The lower panel shows the detailed structureof the chimeric fiber region. The Ad5 fiber tail [amino acids (aa):1–44] were joined in frame to the Ad35 fiber shaft starting from itsfirst two amino acids (GV), which are conserved among many serotypes. Aconserved stretch of amino acids TLWT marks the boundary between thelast β-sheet of Ad35 shaft and the globular knob. The Ad35 fiber chaintermination codon is followed by the Ad5 fiber poly-adenylation signal.

A conserved stretch of amino acids TLWT marks the boundary between thelast β-sheet of Ad35 shaft and the globular knob. The Ad35 fiber chaintermination codon is followed by the Ad5 fiber poly-adenylation signal.

FIG. 19 shows the cross-competition for attachment and internalizationof labeled Ad5GFP, Ad35, and chimeric Ad5GFP/F35 virions with unlabeledviruses, and with anti-CAR or anti-α_(v)-integrins Mab. (A) Forattachment studies, 10⁵ K562 cells were pre-incubated with a 100-foldexcess of unlabeled competitor virus at 4° C. for 1 h. Then, equalamounts of [³H]-labeled viruses, at a dose equivalent to an MOI of 100pfu per cell determined for Ad5GFP, were added to cells followed byincubation at 4° C. for 1 h. Cells were then washed with ice-cold PBS,pelleted and the percentage of attached virus (cell-associated countsper minute) was determined. For analysis of cross-competition forinternalization, cells were pre-incubated with a 100-fold excess ofcompetitor virus at 37° C. for 30 min before labeled virus was added.After an additional incubation at 37° C. for 30 min, cells were treatedwith trypsin-EDTA for 5 min at 37° C., washed with ice-cold PBS,pelleted, and the percentage of internalized virus was determined. Forcontrols, cells were incubated with labeled viruses without anycompetitors. Preliminary experiments had shown that the conditionschosen for competition studies allowed for saturation inattachment/internalization on K562 cells for all unlabeled competitors.(B) 10⁵ K562 cells were pre-incubated for 1 hour at 4° C. with anti-CARMAb (RmcB, diluted 1:100) or with anti-α_(v)-integrin MAb (L230, diluted1:30), followed by incubation with labeled viruses according to theprotocols for attachment or for internalization as described above. Foreach particular serotype, the percentage of attached/internalized viruswas compared to the control settings, where cells were preincubatedunder the same conditions with a 1:100 dilution of an irrelevantantibody (anti-BrdU Mab) before addition of the labeled virus. Note thatthe specific competitors but not the corresponding controlssignificantly inhibited Ad5 internalization to a degree that is inagreement with published data (59). N>/=4. (C) In internalizationstudies, Ad5 did not inhibit internalization of Ad35 or Ad5GFP/F35 intoK562 cells. (D) In internalization studies, L230 monoclonal antibody didnot inhibit internalization of Ad35 or Ad5GFP/F35 into K562 cells.

FIG. 20. Cross-competition for attachment and internalization of[³H]-labeled Ad5GFP, Ad35, and chimeric Ad5GFP/F35 virions withunlabeled Ad3 virus (A), and of [³H]-labeled Ad3 virions with unlabeledviruses (B). 10⁵ K562 cells were pre-incubated with a 100-fold excess ofunlabeled viral particles according to attachment or internalizationprotocols described for FIG. 6. Equal amounts of [³H]-labeled Ad5GFP,Ad5GFP/F35, or Ad35 (A) or [³H]-labeled Ad3 (B) were added to cells at adose equivalent to an MOI of 100 pfu per cell for Ad5GFP. In controlsettings, cells were incubated with labeled viruses without anycompetitors. N=4. (C) In attachment studies, Ad35 does not significantlyinhibit attachment of Ad3 to K562 cells. (D) In internalization studies,cells pre-incubated with Ad35 significantly inhibit internalization ofAd3.

FIG. 21 shows the transduction of CD34+, K562, and HeLa cells withAd5GFP and chimeric Ad5GFP/F35 vectors. 1×10⁵ cells were infected withdifferent MOIs (pfu/cell) of viruses in 100 μl of media for 6 hours at37° C. Virus containing media was then removed, and the cells wereresuspended in fresh media followed by incubation for 18 h at 37° C. Thepercentage of GFP expressing cells was determined by flow cytometry. N=3

FIG. 22 shows the distribution of GFP-positive cells in subpopulationsof human CD34+ cells expressing CAR or α_(v)-integrins. 1×10⁵ CD34+cells were infected with Ad5GFP or Ad5GFP/F35 at an MOI of 200 pfu/cell.Twenty-four hours after infection, cells were incubated with anti-CAR(1:100 final dilution) or anti-α_(v)-integrin (1:30 final dilution)primary MAbs for 1 h at 37° C. Binding of primary antibodies wasdeveloped with anti-mouse IgG-PE labeled secondary MAbs (1:100 finaldilution) at 4° C. for 30 min. For each variant, 10⁴ cells were analyzedby flow-cytometry. The mock infection variants represent cells incubatedwith virus dilution buffer only. The quadrant borders were set based onthe background signals obtained with both the GFP- and PE-matchednegative controls. The percentages of stained cells found in eachquadrant are indicated. The data shown were representative for threeindependent experiments.

FIGS. 23A–23B shows the distribution of GFP-positive cells in asubpopulation of human CD34+ cells, expressing CD34 and CD117 (c-kit).(A) Co-localization of GFP expression with CD34 or CD117: CD34+ cellswere infected with Ad5GFP or Ad5GFP/F35 at an MOI of 200 pfu per cellunder the conditions. Twenty-four hours after infection, cells wereincubated with anti-CD34 PE-conjugated MAbs (final dilution 1:2) or withanti-CD117 PE-conjugated MAbs (final dilution 1:5) for 30 min on ice,and 10⁴ cells per variant were subjected to two-color flow cytometryanalysis. For negative control staining, no antibodies were added to thecells before analysis. The mock infection variants represent cellsincubated with virus dilution buffer only. The quadrant borders were setbased on the background signals obtained with both the GFP- andPE-matched negative controls. The percentages of stained cells found ineach quadrant are indicated. The experiment was performed two times intriplicates, and typically obtained results are shown. The SEM was lessthan 10% of the statistical average. (B) Transduction of CD34+/CD117+cells with Ad5GFP and chimeric Ad5GFP/F35 virus vectors: CD34+ cells,cultured overnight before staining in media without SCF, were incubatedwith PE-labeled anti-CD117 MAb for 30 min on ice. The fraction ofCD117-positive cells was sorted by FACS. More than 97% of sorted cellswere positive for CD117. 1×10⁵ CD117+/CD34+ cells were infected withAd5GFP or Ad5GFP/F35 at an MOI of 200 pfu per cell. Twenty-four hourspost infection, the percentage of GFP positive was determined by flowcytometry. For mock infection, CD117+/CD34+ cells were incubated withvirus dilution buffer only. The infections were done in triplicates, andthe average percentage of GFP-expressing cells is indicated on thecorresponding histogram. The SEM was less than 10% of the statisticalaverage.

FIG. 24 shows the southern analysis of viral genomes in GFP-positive andGFP-negative fractions of CD34+ cells infected with the Ad5GFP andchimeric Ad5GFP/F35 vectors. CD34+ cells were infected with viruses atan MOI of 100 as described for FIG. 21. Twenty four-hours postinfection, cells were sorted by FACS for GFP positive and GFP negativefractions. 10⁵ cells from each fraction were used to isolate genomicDNA, together with viral DNA. Before cell lysis, a rigorous treatmentwith trypsin and DNase followed by washing was performed to exclude thatgenomic DNA samples were contaminated by extracellular viral DNA. A) Theupper panel shows the ethidium bromide stained 1% agarose gel beforeblotting demonstrating that similar amounts of genomic DNA were loaded.This amount corresponded to DNA isolated from ˜25,000 GFP+ or GFP−cells. The lane labeled Aload@ represents viral DNA purified from Ad5GFPor Ad5GFP/F35 virions mixed with pBluescript plasmid DNA (Stratagene) asa carrier and applied on a gel at the amount that was actually used toinfect 25,000 cells. As a concentration standard, a serial dilution ofAd5GFP genomes was loaded on the gel (left side). For Southern analysis(lower panel), an 8 kb-long HindIII fragment corresponding to the E2region of Ad5 was used as a labeled probe. Hybridized filters weresubjected to PhosphoImager analyis and then exposed to Kodak-X-OMAT filmfor 48 h at B70° C. The cellular/viral genomic DNA is indicated by anarrow. (B) To detect Ad5GFP genomes in transduced cells, PCRamplification followed by Southern blot hybridization was performed onthe same samples that were used for quantitative Southern blothybridization in (A). DNA purified from ˜2,500 cells was subjected toPCR (95° CB1 min, 53° C-1 min, 72° CB 1 min, 20 cycles with primersAd5-F1 and Ad5-R1). One fifth of the PCR reaction was subjected toagarose gel electrophoresis (upper panel). A 0.9 kb-long DNA fragment,specific to the E4 region of Ad5 was detected for transduced Ad5GFP/F35genomes. DNA then was blotted onto Nybond-N+ membrane and Southern blothybridization (lower panel) with an Ad5 E4 specific DNA probe wasperformed. In addition to the 0.9 kb DNA fragment, the PCR primersgenerated a smaller 0.5 kb-long fragment that also hybridized with withthe E4 region probe.

FIG. 25 shows the role of fiber shaft length in Ad infection strategies.CAR binding (Ad5 and Ad9) variants and Ad35, which interacts with anon-CAR receptor were analyzed on CAR expressing cells (293, Y79) andK562 cells which do not express significant CAR amounts. All vectorscontain a GFP expression cassette packaged into an Ad5 capsid withmodified fibers.

FIG. 26 shows the tertiary structure of Ad5 knob: localization of CARbinding sites, H—I loop and G-H loop.

FIG. 27 shows the substitution of the G-H loop with heterologouspeptides (SEQ ID NOs.: 14–18).

FIG. 28 shows the attachment and internalization of metabolicallylabeled serotypes with human cell lines.

FIGS. 29A–29D shows the generation of Rep78 expressing Ad vectors byrecombination between two vectors. (A) The same strategy outlined inFIG. 15 was employed for vectors with rep 78 as a transgene. The Ad5′repvector also contained the ApoEhAAT promoter shielded by an HS-4insulator. The region of homology between the two fragments of the rep78gene was 658 nt in length. The Rep78 ORF was deleted for the p5promoter. The internal Rep 40/52 start codon (at position 993) wasmutated to abolish production of the small Rep proteins. Furthermore,the splice site at nt 1905 was deleted eliminating production of Rep68.The individual expression of Rep 78 was demonstrated. (B) Formation ofΔAd.rep78 genomes. The expected 5.8 kb ΔAd.rep78 genome was onlyobserved upon coninfection of both Ad5′rep and Ad3′rep into 293 celss asdemonstrated by Southern. (C) Southern blot analysis for rescue of therecombinant AAV genome from plasmid DNA by Rep78 expressed frompCMVrep78 and ΔAd.rep78. The expected rescue product is 3.8 kb(R-plasmid). (D) Southern blot analysis for rescue of the recombinantAAV genome from Ad.AAV viral vector genomes.

DETAILED DESCRIPTION OF THE INVENTION

All scientific and technical terms used in this application havemeanings commonly used in the art unless otherwise specified. As used inin the present invention, the following words or phrases have themeanings specified.

The term vector includes, but is not limited to, plasmids, cosmids,phagemids, and artificial chromosomes. The vector sequence may bedesignated as the viral “base vector” sequence. The base vector sequenceis dependent upon the particular type of virus and serotype that thebase vector sequence was derived from. The base vector sequence may belinked to non-vector or transgene sequences (e.g., heterologoussequences).

The transgene sequences may include sequences that confer compatibilitywith prokaryote or eukaryote host cells, where compatibility relates tovector replication within a host cell. Accordingly, the transgenesequence may be a replicon sequence that directs replication of thevector within the host cell, resulting in an autonomously replicatingvector. Alternatively, the transgene sequence may permit vectorreplication that is dependent upon the host cell's replicationmachinery.

The base vector sequences may be a operatively linked to a transgenesequence that encodes a gene product, such as a polypeptide, rRNA, ortRNA. For example, the transgene may encode a polypeptide such as aviral capsid protein, or a viral fiber protein. The transgene may bederived from the same or different serotype as the base vector sequence.

Another example of a transgene includes a reporter gene that encodes agene product that can be used as a selectable marker, such as drugresistance or a calorimetric marker. The reporter gene may encode a geneproduct which can be readily detected by, for example, a visualmicroscopic, immunochemical, or enzymatic assay. The preferred reportergene encodes a gene product that can be detected by a non-destructivemethod that does not destroy the cell that expresses the reporter gene.

A therapeutic gene is another example of a transgene. A therapeutic geneencodes a gene product (e.g., polypeptide or RNA) which when expressedin a host cell provides a therapeutic benefit or desired function to thehost cell or the tissue or the organ or the organism containing the hostcell. The therapeutic benefit may result from modifying a functin of agene in the host genome or from the additional function provided by thetherapeutic protein, polypeptide or RNA.

The base vector sequence may be linked to a transgene sequence that isan regulatory element, such as a promoters, enhancers, transcriptiontermination signals, polyadenylation sequences. The regulatory elementmay direct expression of the transgene sequence that encodes a geneproduct by direct transcription or translation. The regulatory elementmay regulate the amount or timing of expression of the transgenesequence. The regulatory element may direct expression of the transgenein certain host cells or tissues (e.g., host-specific or tissue-specificexpression).

The base vector sequence may linked to a transgene sequence that permitsthe vector, to integrate into another nucleotide sequence. Theintegration sequence may direct integration of the whole vector orportions of the vector. The integration sequence may or may not berelated to the base vector sequence. For example the integration andbase vector sequences may be from the same or different viral serotype.The integration sequence may be inverted repeat sequences (ITRs) fromadenovirus (Ad), adenovirus-associated virus (AAV), or HIV.

The base vector sequence may be linked to a transgene sequence thatdirects homologous recombination of the vector into the genome of a hostcell. Such transgene sequences may or may not be from the same viralserotype as the base vector sequence.

The vector may be used to transport the heterologous sequence into ahost cell or into a host cell's genome.

The vector may comprise multiple endonuclease restriction sites thatenable convenient insertion of exogenous DNA sequences.

The term “hybrid vector” as used in the invention refers to a vectorwhich comprises a nucleic acid sequence combined from two differentviruses (e.g. Adenovirus and AAV).

“Chimeric vector” refers to a vector which contains nucleic acidsequences that are unnatural to the base vector (i.e. sequences notoccurring naturally or sequences not in their natural backgroundincluding heterologous sequences). A chimeric vector as used in theinvention may also be a hybrid vector. An example of a chimeric vectoris Ad.AAV expressing a modified fiber protein an its capsid.

The term “transduction” or “infection” refers to a method of introducingviral DNA within a virus particle into a host cell. The viral DNA hereinis in the form of recombinant virus, which is generated by linking asegment of DNA of interest into the viral genome in such a way that thegene can be expressed as a functional protein.

The term “transfection” refers to a method of introducing a DNA fragmentinto a host cell.

The term “heterologous” as used herein means that a nucleic acid orpeptide sequence is placed in a context that is not endogenous to thebase adenovirus vector or to a transduced cell. For example, a peptidesequence can be transferred from a protein to another protein, theresulting protein is referred to herein as heterologous protein. Achimeric fiber protein, (e.g., a serotype 5 tail domain and a serotype35 shaft and knob domain) is considered a “heterologous” to the Ad 5vector. The term also includes nucleic acids (e.g. coding sequences)from one strain or serotype of adenovirus introduced into a differentstrain or serotype of adenovirus.

The term “regulatory elements” is intended to include promoters,enhancers, transcription termination signals, polyadenylation sequences,and other expression control sequences. Regulatory elements referred toin the invention include but are not limited to, those which directexpression of nucleic acid sequence only in certain host cells (e.g.tissue specific regulatory sequences).

The term “operably linked” indicates that a polynucleotide sequence(e.g., a coding sequence or gene) is linked to a regulatory element insuch a way that the regulatory element sequence controls and regulatesthe transcription or translation or both of that polynucleotidesequence. The orientation of the regulatory element may vary (eg, be inreverse orientation with respect to the right ITR). The term alsoincludes having an appropriate start signal (e.g., ATG) in front of thepolynucleotide sequence to be expressed and maintaining the correctreading frame to permit expression of the polynucleotide sequence underthe control of the expression control sequence and production of thedesired polypeptide or protein. Regulatory sequences can also include 3′sequences which ensure correct termination (eg. polyadenylation stopsignal).

The term “gene therapy” used herein, refers to a method which introducesa segment of exogenous nucleic acid into a cell in such a way that itresults in functional modification to the recipient cell by expressionof the exogenous nucleic acid. The exogenous nucleic acid is typicallytherapeutic in that the expression of the encoded protein, polypeptideor RNA corrects cellular dysfunction due to a genetic error or moregenerally counteracts any undesirable functions which are associatedwith a genetic or acquired disease. The term “exogenous nucleic acid”refers to DNA or RNA sequences not normally expressed in the treatedtransformed cell. The term also refers to DNA and RNA sequences whichare expressed in a treated transformed cell at a higher, lower or in anotherwise different pattern than in the untreated, nontransformed cell.This non-natural expression can also be termed heterologous expression.

A “gene therapy vector” refers to a vector used for gene therapy. i.e.to introduce the exogenous nucleic acid into a recipient or host cell.The exogenous nucleic acid may be transiently expressed or integratedand stably expressed in the recipient or host cell.

The term “plasmid” as used herein refers to any nucleic acid moleculewhich replicates independently of the host, maintains a high copynumber, and which can be used as a cloning tool.

The term “parallel strand of DNA” and “anti-parallel strand of DNA”refers to as each of the strands of DNA of the double strandedadenovirus. The Figures diagram the location of certain nucleotides onthe parallel strand of DNA. The anti-parallel strand of DNA refers tothe other of the two strands of DNA which is not depicted in theFigures. The fiber protein is encoded on the anti-parallel strand ofDNA. To simplify the vector diagrams, the fiber sequences are shown onthe parallel strand even though the gene is located on the anti-parallelstrand.

The term “reporter gene” refers to any nucleic acid sequence whichencodes a polypeptide or protein which can be readily detected by, forexample, a visual, microscopic, immunochemical or enzymatic assay.Preferred reporter genes are those that can be detected by anon-destructive method that does not destroy the treated, transformedcells or tissue.

The term “selection gene” used herein refers to any nucleic acidfragment which encodes a polypeptide or protein whose expression is usedto mark a cell as a transformed cell by a given vector.

The term “therapeutic gene” refers herein to a DNA fragment encoding afunctional polypeptide, protein or RNA, which when expressed in a hostcell provides a therapeutic benefit or desired function to the host cellor to the organ or organism containing the host cell. The therapeuticbenefit may result from modification of a function of a native gene in ahost or from the additional function provided by the therapeuticprotein, polypeptide or RNA.

The term “host tissue” or “host cell” as used herein, refers to a tissueor cell in which a therapeutic gene is to be expressed to modify itsfunction.

It is well-known in the biological arts that certain amino acidsubstitutions may be made in protein sequences without affecting thefunction of the protein. Generally, conservative amino acidsubstitutions or substitutions of similar amino acids are toleratedwithout affecting protein function. Similar amino acids can be thosethat are similar in size and/or charge properties, for example,aspartate and glutamate, and isoleucine and valine, are both pairs ofsimilar amino acids. Similarity between amino acid pairs has beenassessed in the art in a number of ways. For example, Dayhoff et al.(1978) in Atlas of Protein Sequence and Structure, Volume 5, Supplement3, Chapter 22, pp. 345–352, which is incorporated by reference herein,provides frequency tables for amino acid substitutions which can beemployed as a measure of amino acid similarity. Dayhoff et al'sfrequency tables are based on comparisons of amino acid sequences forproteins having the same function from a variety of evolutionarilydifferent sources. Therefore, any obvious changes in the amino acidsequences (as described above) to the sequences of the invention arealready contemplated.

Polypeptides which are “substantially similar” share sequences as notedabove except that residue positions which are not identical may differby conservative amino acid changes. Conservative amino acidsubstitutions refer to the interchangeability of residues havingsimilar, side chains. For example, a group of amino acids havingaliphatic side chains of amino acids having aliphatic-hydroxyl sidechains is serine and threonine; a group of amino acids havingamide-containing side chains is asparagine and glutamine; a group ofamino acids having aromatic side chains is phenylalanine, tyrosine, andtryptophan; a group of amino acids having basic side chains is lysine,arginine, and histidine; a group of amino acids having sulfur-containingside chains is cysteine and methionine. Preferred conservative aminoacids substitution groups include but are not limited to:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, asparagine-glutamine, and aspartate-glutamate.Therefore, polypeptide substitution for “substantially similar”sequences (as described above) to the amino acid sequences describedinvention are already contemplated.

In order that the invention herein described may be more fullyunderstood, the following description is set forth.

The present invention provides unique gene transfer vehicles whichovercome many of the limitations of prior art vectors. The inventiondescribes a first generation adenovirus vectors comprising left andright Ad ITRs, an Ad packaging sequence, a transgene cassette withregulatory elements, and a pair of cassette ITRs flanking the transgenecassette that direct predictable viral genomic rearrangements duringviral replication as well as direct the integration of the transgenecassette into the host cell genome.

One predictable rearrangement that occurs during viral replication isthe generation of a gutless adenovirus vector (also referred to hereinas ΔAd) that comprises right and left Ad ITRs, an Ad packaging sequence,a transgene cassette flanked by cassette ITRs and the gutless vector isdevoid of all other immunogenic viral genes.

The potential for site-specific integration is an importantcharacteristic of the novel Ad vectors of the invention. In anembodiment of the invention, integration of the transgene cassette isdirected by co-infection with an Ad vector expressing e.g., the rep 78protein to achieve site-specific integration in the e.g., AAVS1 site onhuman chromosome 19.

The invention further describes a novel way of targeting theserecombinant adenovirus vectors to selected cells by modifying theadenovirus fiber protein that is expressed on the capsid. Changes toboth the fiber shaft and the fiber knob domain proved to successfullyretarget the Ad vector to a desired cell type. In addition, the G-H loopwithin the fiber knob domain is identified as a novel site that affectthe binding affinity and specificity of the recombinant adenovirusvector. Substitution of peptide sequences into the G-H loop retarget thegutless vector to a desired cell type.

An adenovirus display library has been generated that expresses randompeptides within the G-H loop of the fiber protein. This type of alibrary is used as ligands to screen for adenovirus vectors that bind todesired cell types. One advantage of using an adenovirus display libraryversus a phage display library is that once adenovirus affinity to adesired cell is identified the targeted adenovirus vector is ready toaccept a transgene cassette and can be used to generate a gutlessadenovirus vector, for example for use in gene therapy.

The chimeric Ad vectors described below contain a modified fiber proteinin the capsid of the adenovirus which renders the vector capable ofinfecting a desired cell types. Therefore, according to the invention, agutless chimeric ΔAd-AAV vector can be generated to introduce anytransgene(s) into any host cell or tissue which is normally refractoryto most commonly used gene therapy viral vectors. In addition, thechimeric ΔAd.AAV vector of the invention, is devoid of adenoviral genes,and contains AAV ITR sequences that flank the transgene cassette, whichdirect stable transgene integration in the host genome allowing longterm expression of the transgene.

The transgene cassette described in the invention may carry a transgenewhich is either a reporter gene, a selectable gene for in vitro or invivo selection of transduced cells, or a therapeutic gene. In oneembodiment of the invention the reporter trangene can be but is notlimited to, Δgalactosidase. Many reporter genes are commonly used in theart, of which any could be carried as a transgene in the Ad.AAV vectorof the invention. Other examples of reporter are genes are GFP andalkaline phosphatase.

The following describes an embodiment of the first generation Ad vectorsof the invention having a wild-type capsid and a transgene cassetteflanked by cassette ITR sequences; (b) fiber protein that is modified toretarget Ad vectors; and (c) the combination of both technologies thatenables the production of chimeric ΔAd vector including a modified fiberprotein expressed on the capsid which retargets the base vector to adesired cell type for infection and transgene integration.

A. Integrating Ad Hybrid Vectors of the Invention:

It has been shown that inverted repeats (IRs) inserted into the E1region of AdE1-vectors can mediate predictable genomic rearrangementsresulting in a gutless vector genome devoid of all viral genes. Aspecific embodiment of such IR-mediated rearrangements is the Adeno-AAV,first generation hybrid adenovirus vector containing AAV invertedterminal repeats (ITR) flanking a transgene cassette. The AAV ITRsmediate the formation of a genome similar to that of the ΔAd.IR genome(Steinwaerder et al., 2000 Journal of Virology). ΔAd vectors devoid ofall viral genes stably integrate and transduce cultured cells withefficiencies comparable to e.g. rAAV vectors. The Examples demonstrateby Southern blot analysis that the ΔAd vectors integrate randomly intothe host genome.

The Ad vectors of the invention comprise a left Ad ITR, an adenoviruspackaging sequence located 3′ to the Ad ITR; a transgene cassettelocated 3′ to the packaging sequence comprising a polyadenylationsignal, a transgene, and a heterologous promoter, and flanked by a pairof cassette ITRs. Adenoviral genes used for replication such as E1, E2,E3, E4 are located 3′ to the right cassette ITR and a right Ad ITR islocated 3′ to the replication genes. The vectors of the invention areparticularly suited to treat: genetic disorders, cancers, and infectiousdiseases (such as HIV, emboli, or malaria). Treatable genetic diseasessuch as hemophilia A and B; cystic fibrosis; muscular dystrophy, and{overscore (α)}₁ antitrypsin disorder are ideal candidates for geneticdisease that can be treated by vectors of the invention. A specificexample of a therapeutic gene to combat a genetic disorder isgamma-globin to ameliorate sickle cell anemia.

To aid in the selection of transduced cells and characterize theintergration site of the transgene cassette, an embodiment of theinvention includes the addition of a sequence comprising a bacterial forthe origin of replication, plus a selectable gene. An embodiment of thisis an SNori sequence added to the transgene cassette. This allows theΔAd to be expressed in human and bacterial cells, therefore allowingselection of the transduced cells and characterization of theintegration site in the genome of transduced mammalian cells.

The potential for site-specific integration is an importantcharacteristic of the novel Ad vectors of the invention. In anembodiment of the invention, integration of the ΔAd.AAV is directed byco-infection with Ad AAV expressing the rep 78 protein in 293 cells toachieve site-specific integration in the AAVS1 site on human chromosome19. For this type of site-specific integration to occur in cells otherthan 293 cells, E4 ORF6 expression is required. The co-infection ofΔAd.AAV, ΔAd. rep 78, and ΔAd. E4-orf6 allows for site specificintegration of the ΔAd.AAV transgene cassette. The ΔAd. rep78 and theΔAd. E4-orf6 genomes are degraded soon after transduction, thus avoidingpotential side effects. Site-specific integration is preferred overrandom integration, which is seen with rAAV and ΔAd.AAV, in order toreduce the risk of insertional mutagenesis.

Integration of the transgene cassette contained in the adenoviralvectors into chromosomes may be associated with silencing (or blocking)of transgene expression. The silencing of transgenes can be overcome byadding insulator elements to the transgene cassette. For example, HS-4insulator elements derived from the chicken-globin LCR can function inAd vectors to shield heterologous promoters from adenoviral enhancers.HS-4 insulators or the Drosophila Gypsy gene can also be used to preventsilencing transgenes.

Another embodiment of the invention is to split the transgene cassetteinto two portions of the transgene each carried in a differentrecombinant adenoviral vector of the invention. Each portion of the sametrangene has an overlapping region of homology. After infection withboth vectors, each carrying the different but overlapping portion of thesame transgene, homologous recombination event occurs resulting in thereconstitution of the complete transgene which is then expressed. Thistechnique is used to produce hybrid adenoviral vectors that accommodatelarge inserts including, but not limited to a 13 kb genomic hAAT gene ora 12 kb γglobin LCRγglobin expression cassettes for ameliorating sicklecell anemia (or correcting γ-globin mutations). The formation of thehybrid ΔAd vector genomes, after recombination between two vectors, ismore efficient if the overlapping region of homology within thetransgene is longer.

An advantage of the present invention is a method to rapidly isolatepure gutless hybrid adenoviral vectors such as ΔAd.AAV or ΔAd.AAV^(fx)vectors. To minimize the contamination of ΔAd with first generationvectors (Ad vectors) a strategy is described in Example I H. It isanticipated that these approaches will yield the same titer of ΔAdvectors, however the contamination with full-length genome vectors willbe less. This improved isolation of the vectors is extremely importantto avoid toxic side effects after in vivo application.

B. Tropism Modified Adenovirus Vectors:

The Ad vectors of the invention can be modified so that they target ahost cell of interest. There are more than 50 human Ad serotypes(Appendix I), including variants with different tissue selectivity ortropism. It is accepted in the art that different Ad serotypes bind todifferent cellular receptors and use different entry mechanisms. Mostrecombinant adenovirus vectors use adenovirus serotype 5 as the basevector serotype 5 (Ad5) (Hitt, M. M., et all, 1997, Adv. in Pharmacology40, 137–205). Ad5 infection is primarily mediated by its fiber proteinbinding to CAR and secondarily by its penton base protein binding tointegrin. Due to the lack of CAR and/or integrin expression on many celland tissue types, Ad5 mediated gene transfer is inefficient in a numberof tissues which are important targets for gene therapy such asendothelia, smooth muscle, skin epithelia, differentiated airwayepithelia, brain tissue, peripheral blood cells, or bone marrow. Thefollowing describes Ad5 vectors of the invention having a change ininfectivity and tropism as a result of altering the fiber proteinsequence.

The infectivity of different Ad serotypes is limited to a number ofhuman cell lines. Infectivity studies revealed that Ad5 and Ad3 areparticularly suitable for infecting and targeting endothelial orlymphoid cells, whereas Ad9, Ad11 and Ad35 efficiently infected humanbone marrow cells. Therefore, the knob domain of the fiber protein ofAd9, Ad11 and Ad35 are excellent candidates for retargeting the Ad5vector to human bone marrow cells. Other possible serotypes include Ad7.

In the modified fiber protein of the invention the fiber knob domain ofthe Ad5 fiber has been replaced with another Ad serotype fiber knobdomain. An embodiment of the invention is the modified Ad5/35 fiberprotein (a recombinant Ad5 vector expressing a modified fiber proteincomprising of a fiber tail domain of Ad5 and the fiber shaft and knobdomains of Ad35). The Ad5/35 chimeric fiber protein shows a broaderspectrum of infection to a subset of CD34+ cells, including those withstem cell activity. The Ad5/11 chimeric fiber protein (a recombinant Ad5vector expressing a modified fiber protein comprising the fiber taildomain of Ad5 and the fiber shaft and knob domains of Ad11) showedsimilar tropism.

In addition to the knob domain modifications, the invention describesthe added advantage of modifying both the fiber shaft domain and thefiber knob domain to produce a shortened fiber protein. The length ofthe fiber shaft domain plays a key role in the host receptors used forviral vector entry into the host cell. To show this Ad5, Ad5/9, andAd5/35 variants were constructed with long (22β-sheets) andshort-shafted (7β-sheets)-shafted fibers. These analyses demonstratedthat efficient viral infection involving CAR as the primary receptor forAd5, Ad5/9 requires a long-shafted fiber protein, whereas the cell entrystrategy of Ad5/35 (which binds to an still uncharacterized non-CARreceptor) does not depend on the shaft length (FIG. 3). The modificationin both the fiber shaft domain length (between 5>10 β-sheets) and thefiber knob domain (from a different Adserotype than the base vector is anovel mode of altering Ad vector tropism.

To broaden the repertoire of cell types that Ad vectors can infect, aspecific binding region, the G-H loop, within the knob domain has beennewly identified herein to improve binding affinity and specificity.Alteration within this region will redirect the Ad vector to a desiredcell type. For example, the invention describes the G-H loop sequencewithin the fiber protein knob domain, which can be replaced withheterologous peptide ligand sequences without affecting the functionallyimportant tertiary structure of the Ad fiber knob domain, while changingthe binding affinity and specificity of the vector (FIG. 27). This G-Hloop region is exposed on the central part of the knob surface and maybe strategically a better site for incorporation of heterologous ligandsthan the peripheral H-I loop (Krasnykh, V. et al., 1998, J. Virol.,72:1844–52.) of the knob C-terminus (Michael, S. I., et al., 1995, GeneTher., 2:660–8., Wickham, T. J. et al., 1996, Nat. Biotechnol.,14:1570–3.), which are the substitution sites used by others. Therefore,these G-H loop modifications within the fiber knob domain will allow theAd vector to be redirected to infect a desired cell type, as long as theG-H loop ligand sequence binds to at least one surface protein on thedesired cell type. FIG. 27 shows some possible substitutions. Example IIJ demonstrates that the virion tolerates the insertion of a cyclingpeptide (12 amino acids) with a constrained secondary structure thatallows the exposure on the knob surface. A defined ligand (RGD) can beinserted into the G-H and the H-I loop of an Ad5 capsid that is ablatedfor CAR, and integrin tropism. Infectivity studies show the potentialadvantage of this new insertion site.

Use of the Vectors of the Invention for “Gene Therapy”

The liver is the major organ for protein synthesis. Therefore animportant goal of gene therapy is to target gene therapy vectors to theliver. To genetically correct many types of mutant proteins hepatocytesneed to be infected with gene therapy vectors carrying a correctedtransgene. Example II J describes a G-H loop substitution in the knobdomain of the fiber protein with both RI and RII+ (of the malariacircumsporozite surface protein) in a short shafted fiber protein whichdirects the vector to have affinity and specificity to hepatocytes.

Example II K applies a similar protocol to alter the fiber knob domainin the G-H loop region with peptides that target the vector breastcancer cell lines (MDA-MB-435). These novel approaches to redirectvectors described in the invention allow lower doses of the gene therapyvectors to be administered with a higher safety profile.

In example II L a protocol for preparing an adenovirus display libraryis described that uses the fiber knob protein to display a library ofrandom peptide sequences within the G-H loop. This library ofadenoviruses with modified fiber proteins is screened for affinity andspecificity for a desired cell type. There are two main advantages ofusing this adenovirus display library to screen for target peptides thatallow binding to a desired cell type over a phage display librarysystem. First, once a ligand peptide is identified that binds to thedesired cell type it is already in the vector of choice for gene therapydelivery. The peptide does not need to be engineered into anothervector, as is the case for the phage display library vectors. Thisreduces the steps required to identify a targeted fiber protein for adesired cell type. The second advantage of this method, is that theadenoviruses are able to display multiple copies of the modified fiberprotein on their capsid. This allows for dimerization and trimerizationof the fiber protein with the host cell receptor. The multimerization offibers proteins is a realistic, in vivo interaction of the trimericfiber protein with the host cell receptor. In contrast, phage vectorscan only display one fiber peptide sequence on their surface, whichsignificantly limits the ability of interaction with host cell surfacereceptors.

C. A Chimeric Adenovirus Vector with Selective Tropism:

The chimeric vectors of the invention combine two vectors: an Ad.ITR anda Ad. fx where fx describes a modified fiber protein. A first generationadenovirus vector of serotype 5 is the base vector that carries atransgene cassette flanked by heterologous ITRs. These specific invertedterminal repeat sequences, such as AAV ITRs direct stable integration ofthe transgene cassette into the host genome as well as controlpredictable genomic rearrangements that occur during viral replication.This vector can also carry a modified fiber gene (described in ExamplesII). During replication predictable genomic rearrangements occur whichresult in the generation of a gutless adenovirus vector (e.g.ΔAd.AAV^(fx)) which expresses the modified fiber protein on its capsid.The modified fiber protein allows the gutless vector to be targeted to aselected cell type. The targeted vector is a gutless adenovirus vectordevoid of adenoviral genes which can integrate its transgene into thehost genome. The transgene cassette can carry reporter, selectable, ortherapeutic genes.

In one embodiment of the invention, the gutless targeted ΔAd.AAV^(fx)carries the reporter gene of {overscore (Δ)}galactosidaseΔAd.AAV^(fx−)BG). For easy in vitro selection of human and bacterialcells that are transduced with the hybrid Ad vector, a bacterialsequence for the origin of replication can be added to the hybrid Advectors. An example of this is ΔAd.AAV^(fx)-Snori, in which a SNorisequence is added into the transgene cassette. This site allows for G418selection on cells infected with ΔAd.AAV^(fx)-SNori. This in vitroselection provides a tool to analyze the site of transgene integrationand the flanking chromosomal regions. Fluorescent in situ hybridization(FISH) is an alternative method to confirm vector integration.

An advantage of the ΔAd chimeric vector for gene transfer is theefficient and stable integration of a large transgene cassette up toe.g. 22 kb which is significantly larger than the capacity of retroviralvectors. This is of particular interest for gene therapy. For example,to ameliorate sickle cell anemia ΔAd.AAV^(fx){overscore (γ)}globin, anexpression transgene cassette with the gamma-globin gene that targetsand integrates, can be inserted into bone marrow stem cells for longterm expression of the gamma-globin gene.

To achieve site-specific gene integration, rep78 protein is used fortransgene integration into the AAVS1 site (described in Example 1D).However, this may silence transgene expression. To prevent theintegrated transgene from being silenced by host genomic elements (suchas positional effects or downstream enhancers), LCRs or insulatorelements are incorporated into the transgene cassette.

The following examples are presented to illustrate the present inventionand to assist one of ordinary skill in making and using the same. Theexamples are not intended in any way to otherwise limit the scope of theinvention.

EXAMPLE I

Novel Adenoviral Vector Ad.AAV

A. Integrating ΔAd.AAV Hybrid Vectors Devoid of all Adenoviral Genes.

In vitro and in vivo studies with rAAV indicate that the onlyrequirement for rAAV integration are the AAV ITRs and as yet unknownhost cellular factors. It is thought that specific sequences orsecondary structures present in AAV ITRs are prone to integration intohost chromosomal DNA. In order to combine advantages of adenoviralvectors (high titer, high infectivity, large capacity) and theintegration capability of AAV ITRs, AAV vector DNA with AAV ITRsflanking cassettes a secreted human placental alkaline phosphatase(SEAP)-neomycin phosphotransferase (neo) reporter gene cassette(Alexander, I. E., et al. 1996, Gene Therapy, 7, 841–850) isincorporated into the E1-region of E1/E3 deleted adenoviral vectors(Ad.AAV1) (FIG. 1, top).

Methods

Production/Characterization of Viral Vectors

Plasmids:

The AAV1 vector cassette containing AAV ITRs and SEAP/neo expressionunits is obtained by AseI/ScaI digestion of the plasmid pALSAPSN(Alexander, I. E. et al, 1996, Human Gene Therapy, 7:841–50). The 4.4 kbAAV vector fragment was cloned via NotI adapter linkers into pXJCL1(Mirobix, Toronto, Canada) (pAd.AAV1). Another shuttle vector(pAd.AAV1-Δ2ITRs) lacking the AAV ITRs is generated by inserting the 3.7kb AflII/BsmI fragment of pALAPSN into pXJCL1. For pAd.AAV1Δ1ITR, aconstruct is used where a spontaneous deletion in the left AAV ITRsbetween the A and A′ regions has occurred. To create a second hybridvector (Ad.AAV2), the AAVSNori cassette developed by E. Rutledge isused. AAV vector DNA obtained is from pASNori (Rutledge, E. A., Russell,D. W. 1997. Journal of Virology 71:8429–8436) as a 3.4 kb BsaI/ScaIfragment and inserted into the EcoRV site of pXCJL1. As it is generallyknown for AAV vector plasmids, the AAV ITRs are prone to rearrangements.To minimize deletions in these functional critical regions, allconstructs for generation of hybrid vectors are assembled in lowcopy-number plasmids which are grown in E. coli Top10, JC811, or XL1Bluecells (Stragene, La Jolla, Calif.). Furthermore, after each cloningstep or large-scale plasmid amplification, both AAV ITRs are carefullymapped by restriction analysis with enzymes that cut inside or adjacentto the ITRs (BssHII, AhdI, SmaI, Bg1I, BsmI, AflII, and ScaI).

Adenoviruses:

First-generation viruses with the different transgene cassettesincorporated into the E1 region are generated by recombination of thepΔE1aSpla- or pXCJL1-derived shuttle plasmids and pJM17 (Microbix) in293 cells as described earlier (Lieber, A., et al., 1996, J. ofVirology, 70, 8782–8791). For each virus, at least 20 plaques arepicked, amplified, and analyzed by restriction digest. Virusescontaining two AAV ITDRs tend to rearrange within the ITRs, with otheradenoviral sequences, or with adenoviral sequences present in the 293cell genome. Only plaques from viruses with intact ITRs are amplified,CsCl banded, and titered as described earlier (Kay, M. A., et al. 1995.Hepatology 21:815–819; Lieber, A., et al. 1996. Journal of Virology70:8944–8960). All virus preparations tested are negative for RCA andbacterial endotoxin (Lieber, A., et al. 1997. Journal of Virology71:8798–8807). Virus is stored at −80° C. in 10 mM Tris-Cl, pH 7.5–1 mMMgCl₂-10% glycerol.

To generate ΔAd.AAV, 293 cells are infected with Ad.AAV1 at anmultiplicity of infection (MOI) of 25 and harvested 40 h afterinfection. Cells are lysed in PBS by 4 cycles of freeze/thawing. Lysatesare centrifuged to remove cell debris and digested for 30 min at 37° C.with 500 units/ml DNaseI and 200 μg/ml RNaseA in the presence of 10 mMMgCl₂. 5 ml of lysate is layered on a CsCl step gradient (0.5 ml–1.5g/cm³, 2.5 ml–1.35 g/cm³, 4 ml–1.25 g/cm³) and ultracentrifuged for 2 hat 35,000 rpm (rotor SW41). CsCl fractions are collected by puncturingthe tube and are analyzed for viral DNA (Lieber, A., et al. 1996.Journal of Virology 70:8944–8960; Steinwaerder, D. S., et al. 1999. JVirol 73:9303–13) or subjected to ultracentrifugation at 35,000 rpm for18 hours in an equilibrium gradient with 1.32 g/cm³ CsCl. The bandcontaining the deleted viruses ΔAd.AAV is clearly separated (0.5 cmdistance) from other banded viral particles containing full-lengthad.AAV genomes. ΔAd.AAV1 fractions are dialyzed against 10 mM Tris-Cl,pH 7.5–1 mM MgCl₂-10% glycerol and stored at −80° C. The genome titer ofΔAd.AAV1 preparations is determined based on quantitative Southernanalysis of viral DNA purified from viral particles in comparison todifferent concentrations of a 4.4 kb AseI/ScaI fragment of pALSAPSNaccording to a protocol described earlier (Lieber, A., et al., 1996, J.of Virology, 70, 8782–8791). In total, the production of 1×10¹³genome-particles of ΔAd.AAV1 requires less than 3 hours of actual work.

Titers routinely obtained are in the range of 3–8×10¹² genomes per ml.Assuming one genome is packaged per capsid, the genome titer equals theparticle titer. The level of contaminating Ad.AAV1 is less than 0.1% asdetermined by Southern analysis, which is consistent with resultsobtained by plaque assay on 293 cells (fewer than 5 plaques per 10⁶total genomes). The primers used for sequencing the left and rightITR-vector-junction are

5′GGCGTTACTTAAGCTAGAGCTTATCTG (SEQ ID NO.: 1), and

5′CTCTCTAGTTCTAGCCTCGATCTCAC (SEQ ID NO.:2).

SEQ ID NO: 1

SEQ ID NO: 2

The recombinant AAV virus stock containing the SEAP/neo cassette(AV2/ALSAPSN, [Alexander, I. E. et al, 1996, Human Gene Therapy,7:841–50] used in these studies were obtained from Dusty Miller (FHCRC,Seattle). The stock was free of contaminating replication competent AAV(<50 particles/ml) and wildtype adenovirus (<100 particles/ml). Thegenome titer of the virus stock was obtained by quantitative SouthernBlots as described by Russell et al. (Russell, D. et al. 1994 Proc.Natl. Acad, Sci. USA 91:8915–8919).

Electron Microscopy:

For examination of viral particles in the transmission electronmicroscopy studies, CsCl-purified virions are fixed with glutaraldehydeand stained with uranyl acetate as described previously (Lieber, A., etal. 1996. Journal of Virology 70:8944–8960).

Results

During replication of these hybrid vectors in 293 cells, a 5.5 kb genome(ΔAd.AAV1) is efficiently generated and packaged into adenovirus (Ad5)capsids. The ΔAd.AAV1 genome contains the left adenovirus ITR and thepackaging signal followed by the AAV-vector cassette and a duplicate ofthe adenoviral packaging signal and ITR in reverse orientation (FIG. 1,bottom). The hybrid vector is devoid of all viral genes, thuseliminating toxic effects and the elicitation of cellular immuneresponses. The spontaneous formation of the small hybrid vector genomeΔAd.AAV1 requires the presence of two intact AAV ITRs and does not occurwith partly deleted ITRs or oligo-dC and oligo-dG stretches flanking theexpression cassette.

Hybrid Vectors Containing Different Transgenes:

To construct a hybrid vector with a transgene that can be detected insitu the SEAP/neo expression unit in Ad.AAV1 is replaced by the E. coliβ-galactosidase gene. This hybrid vector is named ΔAd.AAV1. Duringgeneration of the corresponding plasmid constructs the AAV ITR sequencestend to rearrange and abolish their functional properties. This problemcan be circumvented by using low copy number plasmids as cloning vectorsgrown in bacteria strains depleted for all recombination proteins (e.g.JC811). Furthermore, the intactness of both AAV ITRs after each cloningstep can be examined for characteristic endonuclease digestion.Recently, another hybrid vector ΔAd.AAV1Nori has been generated whichcontains the neo gene under the control of both the simian virus 40(SV40) early promoter and the transposon 5 (Tn5) promoter for expressionin human and bacterial cells, as well as the p15A bacterial replicationorigin with the direction of the leading strand DNA synthesis oppositethat of neo gene transcription. Thus, SNori can be used for, G418selection of integrated vector in eukaryotic cells as well as for rescueof vector together with flanking host DNA after integration. Therecovered plasmids can be propagated in E. coli under selection withkanamycin due to the bacterial origin and the neo gene. SNori containingvectors allow a rapid estimation of total integration events based onthe number of G418 resistant colonies. Moreover, vector DNA togetherwith flanking chromosomal DNA can be rescued as plasmids from singleG418 resistant clones and can be used for sequencing to determineintegration junctions. Both hybrid vectors are produced at a titer ofabout 3×10¹² genomes per ml. The ratio of genome titer to transducingparticles for ΔAd.AAVBG is ˜200:1 based on β-Gal expression.

Discussion

ΔAd.AAV1 could spontaneously form during adenovirus replication. Anotherpossible mechanism of ΔAd.AAV1 formation is based on the uniquemechanism by which adenovirus replicates its genome (van der Vliet, B.,1995, In w. Doerfler, et al. (eds.) vol. 2 p. 1–31, Springer-Verlag,Berlin) (see FIG. 1). Ad DNA replication is initiated by the TP/pTP(terminal protein) that binds to specific sites within the ITRs on bothends of the linear genome and catalyzes, in complex with Ad pol, thebinding of the 5′ CTP, the first nucleotide of the daughter strand. DNAsynthesis proceeds in a continuous fashion to the other end of thegenome (FIG. 1A). Only one of the DNA strands serves as template. One ofthe replication products is a single-stranded DNA that circularizesthrough annealing of its self-complementary ITRs. The resulting duplex“panhandle” has the same structure as the termini of the duplex viralgenome that allows the binding of pTP and the initiation for synthesisof a complementary strand using the single-stranded “pandhandle”molecule as template (FIG. 1C). In the case of Ad.AAV1, the Ad polsynthesizes the single strand of the adenoviral genome starting from theleft Ad ITR until it reaches the second AAV ITR. During synthesis of thesecond AAV ITR a certain percentage of the single-stranded moleculesform a loop hybridizing to the complementary region within the first AAVITR that was replicated earlier, allowing Ad pol to use the same viralDNA strand to read back towards the left ITR (FIG. 1B). The resulting“panhandle” structure can be resolved in a similar way as a full-lengthintermediate shown in FIG. 1C, generating a double stranded, linearmolecule with the above described structure that can be packaged into Advirions. The ratio of viral DNA to protein concentration in purifiedΔAd.AAV1 particles is comparable to that obtained from Ad.AAV1particles. This indicates that despite the smaller size, only oneΔAd.AAV1 genome is packaged, resulting in particles with a lighterbuoyant density (˜1.32 g/cm³). Electron microscopy demonstrates theicosahedral shape of ΔAd.AAV1 particles (FIG. 2). Staining with uranylacetate causes the central viral cores to appear electron dense.ΔAd.AAV1 virions have only a spotted luminal dark staining as expectedwith only one 5.5 kb genome being packaged per capsid.

B. In Vitro ΔAd.AAV1 Production:

Characteristics of Deleted adeno-AAV Vectors (ΔAd.AAV):

A number of experiments to clarify the mechanisms of ΔAd.AAV genomeformation are carried out. Specifically, the presence of two intact AAVITRs flanking a reporter gene cassette is required for the effectiveformation of ΔAd.AAV genomes. This process does not occur with partiallydeleted ITRs or oligo-dC and oligo-dG stretches flanking the expressioncassette. Furthermore, in vitro transduction studies are performed withdifferent genome titers of ΔAd.AAV1, Ad.AAV1, and Ad.AAV1-Δ2ITRs,(lacking the two AAV ITRs) which determine the number of G418 resistantcolonies that formed after 4 weeks of selection (Table I).

ΔAd.AAV1 is routinely produced at a high titer (5×10¹² genomes per mlwith >10⁴ produced genomes per 293 cell) and at a high purity with lessthan 0.1% contaminating full length Ad.AAV1 genomes by a techniquenormally used for amplification and purification of recombinantadenovirus.

In Vitro Transduction Studies with Hybrid Vectors on CD34+ Cells andErythroleukemia Cells:

In order to test whether the hybrid vectors allow for gene transfer intocell types, that have to be targeted for sickle cell therapy,infection/transduction studies are performed using CD34+enriched humanbone marrow cells, derived from mobilized peripheral blood and the humanerythroleukemia cell line K562 which express ε and γ globin genes.

Methods

Cell Culture:

SKHep1 cells (HTB-52, American Type Culture Collection, Rockville, Md.),an endothelial cell line derived from human liver [Heffelfinger, S. C.,et al., 1992, In vitro Cell Dev. Biol. 28A, 136-4-142], are grown inhigh-glucose Dulbecco's modified Eagle medium with 10% fetal calf serum.SKHep1 cells are analyzed for integrated AAV provirus by Southernanalysis of genomic DNA using the AAV1 wild type genome obtained frompAAV/Ad (Samulski, R. J., et al. 1989. Journal of Virology 63:3822–3928)(gift from David Russell, University of Washington) as a probe. Nospecific bands are detected in undigested genomic SKHep1 DNA or afterdigestion with HindIII. For viral infection, confluent cells areincubated with different viral doses for 2 hours, followed by intensivewashing. For G418 selection, 24 h after infection with ΔAd.AAV1, SKHep1cells are trypsinized and plated at different dilutions under G418selection (900 μg/ml active compound, Boehringer-Mannheim, Germany).G418 containing culture medium is changed every 3 days. The number ofcolonies with >1 cells is counted after 4 weeks of selection and dividedby the number of initially seeded cells. This ratio is used to expressthe integration frequency of ΔAd.AAV1. Single colones are obtained bylimiting dilutions of infected cells in 96 well plates. Colonies areexpanded to 1×10⁶ cells in the presence of G418. Immunofluorescenceanalysis for adenoviral proteins expressed in SKHep1 cells 3 dayspost-infection is performed as described earlier [Lieber, A., et al.,1996, J. of Virology, 70, 8782–8791].

Results

293 cells are infected with the first generation vector Ad.AAV1. Duringreplication of Ad.AAV1, the small ΔAd.AAV1 genome forms spontaneouslyand is packaged into adenovirus capsid. At 36 hours after infectioncells are harvested and virus is released by several cycles offreeze/thawing. The mixture of Ad.AAV1 and ΔAd.AAV1 particles in thecell lysate is then separated by ultracentrifugation in a CsCl stepgradient. Due to its lighter buoyant density, the band containing theΔAd.AAV1 particles is clearly separated (0.8 cm distance) from the bandcontaining full-length virus (Lieber, A., et al. 1999. J Virol73:9314–24). ΔAd.AAV1 is purified further by an additional CsClequilibrium gradient and is stored in 10 mM Tris pH7.5, 10% glycerol, 1mM MgCl₂ in 80° C. In total, the production of 2×10¹³ (genome) particlesof ΔAd.AAV1 requires less than 3 hours of work. All functions forΔAd.AAV1 replication and particle formation are provided from Ad.AAV1genomes amplified in the same cell. The efficiency of vector productionmeasured on a genome-per-cell-basis is comparable or higher thanlabor-intensive, newer techniques for rAAV production, which have notyet been proven to be reliable. The estimated ratio oftransducing/genome titer for ΔAd.AAV1 is 1:200 (based on SEAP expressionat day 3 post-infection), whereas for the average rAAV preparation, itis in the range of 1:10³ to 1:10⁴. 1×10⁵ confluent SKHep1 cells areinfected with different MOIs of rAAV1 (stock: 1×10¹⁰ genomes per ml),ΔAd.AAV1 (stock: 5×10¹² genomes per ml), Ad.ADAV1 (stock: 1×10¹³ genomesper ml), and AdAAV1 2ITR (stock: 9×10¹² genomes per ml), in a volume of100 ml 24 hours after infection, cells are washed, trypsinized, andplated at different dilutions. G418 is added 24 hours after plating andselection is performed for 4 weeks. G418 resistant colonies contain onaverage >5×10⁴ cells (at least 16 cell divisions). A significant numberof small colonies visible at 2 weeks post-infection do not survivecontinued selection, probably due to episomal vector expression. Cellsinfected with first-generation adenoviruses with MOIs greater than 1×10⁴develop CPE during the first week of selection. The rAAV titer is nothigh enough to perform infection studies with MOIs greater than 10⁴. Thecolony formation is expressed as percentage of the number of coloniesafter selection to the number of cells initially seeded for selection(Table I).

TABLE I Formation of G418 resistant colonies after infection with hybridviruses in comparison with rAAV. Formation of G418 resistant colonies in% (SEM) MOI (after 4 weeks of selection) (genomes Ad.AAV1 per cell)rAAV1 Ad.AAV1 Ad.AAV1 2ITRs 10¹ 0  0 0 0 10² 0  0 0 0 10³ 2.7  1.3 5.4 0(1.6) (1) (3.0) 10⁴ 90.8  48.0 12.9 0 (7.0) (8.9) (7.2) 10⁵ N/A  93.13.8 0 (5.4) (2.1) 10⁶ N/A 100 0 0 10⁷ N/A 100 0 0 N = 3 (SEM isindicated in parentheses.)K562 Cells are Infected with Different MOIs of ΔAd.AAVBG (1–10⁸ Genomesper Cell):

Three days after infection, the total number of viable cells (based onTrypan blue staining) and the percentage of infected cells (based onX-Gal staining) are determined for all MOIs. The results are presentedin FIG. 4A.

Initial Integration Studies:

K562 cells are incubated with ΔAd.AAVSNori at an MOI of 2×10⁵ genomesper cell and the colonies that formed after 4 weeks of G418 selectionare counted in 96 well plates. G418 resistant colonies contain onaverage >5×10⁴ cells which means that the original cell underwent atleast 16 cell divisions.

Infection Studies with Ad.AAVBG (1–10⁸ Genomes per Cell) on CD34+ Cells:

Cell infection on CD34+ are as described for K562 cells. CD34+ cells arecultured in IMDM supplemented with 20% FCS, kit ligand (stem cellfactor-SCF) (100 ng/ml), and IL-3 (100 ng/ml). Since a number of reportssuggest that specific cytokines like GM-CSF or M-CSF which induce stemcell differentiation can stimulate integrin expression and may thereforeaffect internalization of Ad5 vectors, infection rates are compared withAd5 based hybrid vectors on CD34+ cells cultured with and withoutpre-stimulation with GM-CSF (50 ng/ml) or M-CSF (50 U/ml). The number ofinfected cells is counted based on X-Gal staining at day 3 afterinfection. To test for dose-dependent toxicity, viable cells are counted(based on trypan blue exclusion) at day 3 post-infection. Furthermore,whether high viral doses affect the ability of CD34+ cells todifferentiate in methyl cellulose colony assays in presence of IL-3 andSCF is analyzed. The results are expressed as viable cells/X-Galpositive cells vs MOI (see FIG. 4B).

Discussion

The above data demonstrate that ΔAd.AAV transduces stably animmortalized human cell line with a low frequency comparable to rAAV,however, transduction rates could be scaled up to 100% by using greaterMOIs of ΔAd.AAV1, which is produced at higher titers than rAAV1. Incontrast to infection with the first-generation vector, Ad.AAV1,infection with ΔAd.AAV1 is not associated with dose-dependentcytotoxicity because no viral proteins are expressed from these vectorsin transduced cells. Furthermore, viral proteins present in the incomingΔAd.AAV1 particles are not problematic in the dose range use. Thecomparison of transduction rates of ΔAd.AAV/Ad.AAV1 with the vectorlacking AAV ITRs, Ad.AAV-Δ2ITRs, supports the hypothesis that thepresence of two intact AAV ITRs is crucial for hybrid vectorintegration.

The data demonstrate that the leukemia cell line can be infected at ˜90%efficiency with Ad5 based hybrid vectors at MOIs 2×10⁵ genomes per cellwithout significant toxic side effects. However, this dose is still ˜100times greater than the dose necessary to infect 100% of HeLa cell,hepastoma cells, primary hepatocytes and other cell lines generallyconsidered as permissive for Ad5 vector infection.

Since viral DNA in cells infected with 2×10⁴ genomes (or 100 transducingparticles per cell) should be lost after 7 cell divisions, the presenceof G418 resistant cells in the observed colonies suggests thatΔAd.AAVSNori genomes are integrated into or stably associated with thehost genome. Based on the number of G418 resistant colonies one out of25,000 ΔAd.AAVSNori genomes integrates stably into K562 cells. This isin agreement with the results obtained earlier with ΔAd.AAV1 in SK Hep1cells.

The maximal dose used for infection of CD34+ cells (1×10⁸) results inX-Gal staining of only ˜10% of cells independently of GM-CSF/M-CSF. Thisdemonstrates the obvious inability of Ad5 to infect CD34+ cells and isprobably caused by the absence of specific receptors and/or integrins onthe cell surface. CD34+ cells tolerate a large range of viral doses(1–10⁷) without obvious effects on cell viability and total cell number.This is not surprising because in order to develop toxic side effectsadenovirus has to enter the cell and express viral genes. Hybrid vectorscan be produced at titers of 5×10¹² genomes per ml. Thus, the maximalMOI that can be used for infection (of 10⁴ cells) is ˜5×10⁸ (in 100 μlstorage buffer). Based on the infection studies with ΔAd.AAVBG this dosemay not be sufficient to efficiently transduce CD34+ cells and to obtainan appreciable number of G418 resistant colonies.

C. In Vivo Properties of ΔAd.AAV1:

Viral DNA is labeled with BrdU during virus amplification to investigatecellular/nuclear vector uptake in situ. For transduction studies,confluent SKHep1 cells (a human endothelial cell line) are infected with2000 genomes ΔAd.AAV1 or Ad.AAV1 per cell. BrdU tagged viral DNA isdetected in 100% of nuclei at 3 hours post-infection for both virusesindicating efficient cellular and nuclear uptake of hybrid virus DNA.

Results

The ΔAd.AAV1 vector transduces a cell in vitro forming G418 resistantcolonies with an efficiency of 17 or 58%, after infection with an MOI of1×10³ or 1×10⁴ genomes per cell, respectively. Approximately 2×10⁴ΔAd.AAV1 genomes are required to yield one stable transfectant. Sinceall stable colonies contain integrated ΔAd.AAV1 vector DNA, this numberreflects the minimal integration frequency of ΔAd.AAV1 in SKHep1 cellswhich is comparable with that from rAAV (Rutledge, E. A. et al., 1997,Journal of Virology, 71:8429–36). The number of G418 resistant coloniesdoes not necessarily represent the total frequency of integration eventsbecause not all integrated copies express neomycin phosphotransferase,due to chromosomal position effects or incomplete integration.

The absence of adenoviral gene products in ΔAd.AAV1 transduced cells atday 3 post-infection is demonstrated by immunofluorescence withantibodies to the major late proteins (hexon, fiber) and early proteins(DBP.E4-orf6). Expressed adenoviral proteins are detected only in cellsinfected with Ad.AAV1. The fact that cells infected with ≢Ad.AAV1 do notexpress potentially cytotoxic adenoviral proteins is important.

While an MOI of 1×10⁴ genomes per cell of the first generation vectorAd.AAV1 induce cytopathic effects in SKHep1 cells at day 3 p.i., notoxic side effects are observed when SKHep1 cells are infected withΔAd.AAV1 at a dose of up to 1×10⁸ genomes per cell. Since thetransduction efficiency is clearly dose dependent, ΔAd.AAV1 (which canbe produced at titers of >5×10¹² genomes/ml) is able to stably transduceAd5 permissive cell lines or tissues with a 100% efficiency withoutassociated toxicity.

Southern analysis indicates that ΔAd.AAV1 integrates randomly ashead-to-tail tandem repeats into the host cell genome via the right AAVITR, whereas the other junction with the chromosomal DNA is variable andoccurs somewhere within the transgene cassette. In order to confirm theintegrated status of ΔAd.AAV1 DNA, high-molecular-weight chromosomal DNAis separated by pulse field gel electrophoresis (PFGE), followed bySouthern analysis with a SEAP specific probe (FIG. 3). Undigested DNAfrom control SKHep1 cells give an endogenous SEAP signal thatco-migrates with chromosomal DNA just below the well (lanes 1 and 5). Nohigh-molecular weight episomal forms of ΔAd.AAV1 DNA are detected,whereas a distinct 35 kb band is visible in DNA from SKHep1 cellsisolated 3 days after infection with first generation adenovirus,Ad.AAV1 (lanes 4 and 13). Digestion with EcoRI reveals the 4.4 kbfragment, which is specific for integrated tandem copies of the AAVcassette (lanes 8 and 12). To eliminate the possibility that chromosomalDNA is trapped in the well, DNA samples are digested with intron-encodedendonucleases PI-Sce1 or I-CeuI (Gibco-BRL, Grand Island, N.Y.) with asequence specificity or more than 11 bp or 9 bp respectively. Digestionwith PI-SceI yields a >2 mb endogenous SEAP signal in SKHep1 cells (lane2) and an additional signal in the range of ˜1 mb in G418 resistantcolonies transduced with ΔAd.AAV1 (lane 7). I-CeuI digestion results ina smear between 250–1000 kb in ΔAd.AAV1 transduced SKHep1-cells (lanes10, 11) indicating random integration, whereas a high-molecular weightband specific for the endogenous SEAP gene is observed in control SKHep1cells (lane 9).

One day after intraportal infusion of 1×10¹² ΔAAd.AAV1 genomes inC57B1/6 mice, BrdU labeled vector genomes can be detected in 85%hepatocytes (Lieber, A., et al. 1999. J Virol 73:9314–24).Hepatocellular DNA analysis performed at 2 months post-infusion revealsΔAd.AAV1 DNA integrated with an average of 0.5 copies per cell into themouse genome (Lieber, A., et al. 1999. J Virol 73:9314–24). To assesspotential side effects of intraportal ΔAd.AAV1 infusion, serum glutamicpyruvic transaminase (SGPT), a sensitive marker for hepatocellularinjury, is measured for 7 consecutive days post-infusion in combinationwith histological analysis of liver sections. No significant elevationin SGPT levels, or histological abnormalities are detected afterintraportal infusion of 1×10¹² or 1×10¹³ ΔAd.AAV1 genomes, whereasinfusion of the same dose of full-length Ad.AAV1 vector is associatedwith severe hepatoxicity or fatal outcome. This suggests that the doseof ΔAd.AAV1 administered to mice can be increased to obtain highertransduction efficiencies in vivo without adverse side effects, which isnot possible for first generation adenoviruses. Importantly, ΔAd.AAV1transduced quiescent hepatocytes in vivo, which suggests thatintegration of hybrid vector DNA may not require cell proliferation.Recently, more detailed in vivo transduction studies with Ad.AAV1 andΔAd.AAV1 have been performed in Balb/c mice to study whether the absenceof adenoviral gene expression in cells infected with ΔAd.AAV1 can avoidan anti-viral immune response and can prolong vector persistence. Inthis mouse strain, vector DNA is cleared from the liver at 4–6 weeksafter infusion with first generation adenoviruses, mostly due to a CTLresponse against viral proteins produced in transduced cells. Vector DNAis analyzed by genomic Southern Blot of hepatic DNA at 12 weeks afterinfusion of 1×10¹² genomes Ad.AAV1 or ΔAd.AAV1. At this time point, novector specific signal is detectable in hepatic DNA from mice infusedwith the first generation vector Ad.AAV1, while ˜0.3 copies of ΔAd.AAV1genomes per cell are present in livers of mice that received the hybridvector, again indicating the superior in vivo properties of the hybridvector.

D. Effects of Rep Coexpression on ΔAd.AAV1 Integration

Rep Expression after Plasmid Transfection:

In order to test whether Rep expression enhances site-specificintegration of ΔAd.AAV1 in human cells, a series of Rep expressionplasmids are constructed.

Methods

The Rep ORF 68/78 (nt 285–2313) including the internal p19 and p40promoters is obtained from pAAV/Ad (Samulski, R. J. et al., 1991, In B.N. Fields, et al. (eds.), Fields Virology, vol. 2 Lippincott-RavenPublisher, Philadelphia) by digestion with BsaI/BsrI. This fragmentdeleted for the AAV p5 promoter is cloned via adapter linkers under RSVor PGK promoter in front of the bovine growth hormone polyadenylationsignal (bPA) into pAd.RSV or pAd.PGK (Lieber, A., and Kay, M. A., 1996,J. of Virology, 70, 3153–3158; Lieber, A., et al., 1995, Human GeneTherapy, 6, 5–11) correspondingly.

Results

The resulting plasmids (pRSVrep, pPGKrep) are transfected into 293 cellsor SKHep1 cells, most of the Rep proteins expressed from theheterologous promoters (RSV or PGK) are Rep 68 and Rep 78, whiletransfection of the rep gene under aP5 promoter (pAAV/Ad) results inpredominant Rep 52/40 expression. Thus, transfection of pRSVrep andpPGKrep is more pronounced suggesting a strong transactivation of AAVpromoters by E1a which is produced in 293 cells. This result indicatesthat minimum expression of rep proteins is necessary to avoidinterference with adenovirus replication.

Rep-Mediated Site-Specific Integration of ΔAd.AAV1.

The potential for site-specific integration is an importantcharacteristic of the novel Ad.AAV vectors of the invention. In anembodiment of the invention, integration of the ΔAd.AAV is directed byco-infection with Ad AAV expressing the rep 78 protein to achievesite-specific integration in the AAVS1 site on human chromosome 19. Forthis type of site-specific integration to occur in cells other than 293cells, E4 ORF6 expression is required. The co-infection of ΔAd.AAV, ΔAd.rep 78, and ΔAd. E4-orf6 allows for site specific integration of theΔAd.AAV transgene cassette. The ΔAd. rep78 and the ΔAd. E4-orf6 genomesare degraded soon after transduction, thus avoiding potential sideeffects. Site-specific integration is preferred over random integration,which is seen with rAAV and ΔAd.AAV, in order to reduce the risk ofinsertional mutagenesis.

A preliminary test can be performed to confirm the functional activityof Rep 68/78 expressed from pRSVrep to mediate site-specific integrationof ΔAd.AAV1 (FIGS. 5 and 6). Human SKHep1 cells are transfected withpRSVrep or control plasmid (pRSVbGal (Lieber, A., et al., 1995, HumanGene Therapy, 6, 5–11) (transfection efficiency was ˜20%), followed byinfection with ΔAd.AAV (2000 genomes per cell). Three days afterinfection, cells are trypsinized, embedded in agarose, lysed in situ,digested with I-CeuI (an intron-encoded endonuclease with a recognitionsequence of more than 10 nt), subjected to pulse file gelelectrophoresis in 1% agarose gel, and analyzed by Southern Blot.Hybridization with a probe covering the AAVS1 integration site (1.7 kbEcoRI/BamHI fragment from the chromosome 19 locus (Samulski, R. J. etal., 1991, In B. N. Fields, et al. (eds.), Fields Virology, vol. 2Lippincott-Raven Publisher, Philadelphia)) reveals an AAVS1-specificband (˜240 kb) in I-CeuI digested DNA from cells after control plasmidtransfection (pCo)+ΔAd.AAV1 infection. An additional signal in the rangeof 280 kb appears in rep expressing cells infected with ΔAd.AAV1(pRSVrep+VAd.AAV1) indicating a site-specific insertion into the AAVS1site in a certain percentage of cells. The presence of vector DNA inthis 280 kb band is confirmed by rehybridization of the same filter witha transgene (SEAP) specific probe. Randomly integrated ΔAd.AAV1 vectorappears as a diffuse SEAP signal in the range 280–680 kb (pCo+ΔAd.AAV1,pRSVrep+ΔAd.AAV1). The specific ˜1.9 mb band on blots hybridized withthe SEAP probe represents an I-CeuI fragment containing the endogenoushuman SEAP gene.

Incorporation Rep 68/78 function into hybrid vectors to stimulatesite-specific integration Rep overexpression inhibits adenovirus DNAreplication, prohibiting the generation of rep expressing Ad vectorsusing conventional strategies. To solve this problem, significant Rep68/78 expression from the hybrid vector in virus producer (293) cellsmust be prevented while maintaining transient Rep expression in targetcells (HSC) to mediate site-specific integration. Our hypothesis is thatthe specific structure of the ΔAd.AAV hybrid virus can be used to bringthe rep gene 68/78 into a transcriptionally active position undercontrol of a HSC specific promoter only at late stages of virusreplication in 293 cells. This will allow amplification of the hybridvector in 293 cells, generating high titer virus which activates theincorporated Rep 68/78 functions only in HSC. The general outline of ourstrategy to produce Rep expressing hybrid vectors is illustrated in FIG.7. The rep/transgene cassette is assembled based on the left-handshuttle plasmid used for recombinant adenovirus production. The geneencoding Rep 68/78 is cloned in 3′□5′ orientation in front of atransgene expression cassette flanked by AAV ITRs. Between the transgenecassette and the right AAV ITR an HSC-specific promoter is inserted withdirection towards the adenoviral E2, E3, and E4 genes. The recombinantgenome is produced by recombination in E. coli and transfection into 293cells generates virus (Ad.AAV-rep). The specific structure of ΔAd.AAVwith duplicated sequences flanking the AV ITRs is used to bring the repgene into a transcriptionally active position under control of a HSCspecific promoter only during late stages of viral DNA replication in293 cells. During amplification of Ad.AAV-rep, the smaller genomeΔAd.AAV-rep is formed and packaged into particles, which can beseparated by ultracentrifugation in CsCl gradients. The specificstructure of ΔAd.AAV-rep brings the rep gene into 5′

3′ orientation in relation to the HSC specific promoter, allowing reptranscription in target cells. After transduction of HSC with purifiedΔAd.AAV-rep particles, rep expression is activated and mediates rescueof the AAV-ITR/transgene cassette from the adenoviral vector backboneand site-specific integration. The hypothesis is that Rep-mediatedintegration into AAVS1 occurs via the right or both AAV/ITRs causing therep gene to become separated from the hepatocyte-specific promoter oncethe vector is integrated (FIG. 7). Therefore, rep expression should beonly transient without critical cytotoxic side effects on the host cell.

Promoters that can Regulate Rep Expression:

Potential candidate promoters to drive rep expression with highspecificity for HSC and minimal activity in 293 cells are the 454 ntCD34 promoter (Krause, D. S., et al., 1997, Experimental Hemotology, 25,1051–1061; Yamaguchia, Y. et al., 1997, Biochimica et Biophysica Acta.,1350:141–6), the 300 nt HS 40 enhancer (Chen, H. L., et al., 1997,Nucleic Acids Res. 25, 2917–2922) or a 3 kb CD34 enhancer (May, G. etal., 1995, EMBO J., 14:564–74) in combination with an initiator, or theHIV LTR. An optimal promoter is selected based on studies of transientreporter gene expression after plasmid transfection in 293 cells andhepatocytes. All promoters to be tested are cloned in front of the humanα₁-antitrypsin (hAAT)-bovine growth hormone polyadenylation signal (bPA)into the adenoviral shuttle plasmid pCD2 (pAd.-hAAT). Promoter activitycan be tested in transient plasmid transfection assays in CD34+ and 293cells. The promoter with the highest hAAT levels in CD34+ or K562 cellsand the lowest hAAT expression in 293 cells is selected for furtherstudies. If high background expression in 293 cells from these promotersis seen, insulators to shield HSC-specific promoters from the Elaenhancer which is still present in Ad shuttle plasmids can be utilized.

Rep Genes:

The large Rep 68/78 proteins are sufficient to mediate rescue andsite-specific integration. Unregulated Rep 52 and Rep 40 expression fromthe AAV p19 promoter located within the ORF of Rep 68 and 78 must beprevented because production of these smaller Rep proteins in 293 cellswill affect cell viability and adenoviral DNA synthesis. To do this,constructs obtained from Surosky et al., containing a mutated Rep 52/40start codon to express Rep 68 and 78 individually under CMV promoter canbe used. The 293 cells transiently expressing Rep68 or Rep 78 from theseconstructs can be coinfected with ΔAd.AAV1 (infection 24 hours afterpCMVRep transfection, MOI 2×10⁵ genomes/cell). Three days after ΔAd.AAV1infection, cellular DNA is analyzed for AAVS1-specific integrationevents by PCR and PFGE as described earlier. Efficient Rep mediatedexcision of the AAV cassette and site-specific integration withoutflanking adenoviral sequences are expected and the plasmids pCMVRep68 orpCMVRep78 can be used as a source for the corresponding rep genes andclone them into hybrid vectors.

Vectors:

The rep/transgene cassette can be assembled based on pXCJL (Microbix,Toronto). A set of control hybrid vectors can be generated with theAAV-ITR-transgene cassette only without the rep gene. The recombinantAd.AAV-rep genome can be generated by recombination of the left handshuttle plasmids with pCD1, a pBHG10 (Microbix, Toronto) derivative,which contains the Ad5 genome deleted for the E1/E3 regions in recA⁺ E.coli (Chartier, C., et al., 1996, J. of Virology, 70, 4805–4810).Compared to the standard technique based on plasmid recombination in 293cells, this approach has the advantage that plaques with recombinantvirus appear 3 times faster and the production of illegitimaterecombinants is minimized. This allows efficient viral DNA amplificationand packaging to occur before Rep expression reaches levels that arepotentially inhibitory for adenoviral replication. The criticalvariables in maximizing the output of the vector deleted for alladenoviral viral genes are the initial multiplicity of infection and thetime of harvesting. These parameters can be optimized for production ofΔAd.AAV-rep hybrid vectors. A number of ΔAd.AAV vectors can beconstructed incorporating rep gene. Cryptic promoter and enhancerelements present in the 5′-342 nt of the adenoviral genome can interferewith transgene expression from the heterologous promoters. This iscrucial for the strategy to avoid rep expression from ΔAd.AAV-repgenomes in 293 cells. To ensure efficient transgene expression,insulator fragments such as the chicken beta-globin insulator can beused with a selected promoter, constitutive or inducible.

Rep Protein Co-Packaging:

As an alternative to producing hybrid vectors containing the rep 68/78gene, studies are designed to see whether Rep protein can be co-packagedinto ΔAd.AAV capsids and whether these co-packaged Rep molecules aresufficient to mediate rescue and site-specific integration of theAAV-ITR-transgene cassette. Our hypothesis is that the Rep 68/78 bindsto the Rep binding site (RBS) present in double-stranded ΔAd.AAV genomeand that this complex is co-packaged into adenoviral capsids which arespacious enough to accommodate extra proteins. Based on protein/DNAratio analysis performed previously in purified particles that only one5.5 kb ΔAd.AAV1 genome is packaged per capsid. This is confirmed byelectron-microscopy of ΔAd.AAV1 particles, which reveals only spottedelectron-dense staining associated with viral cores and extended freeluminal space (see FIG. 2).

293 cells are transfected with plasmids expressing Rep 68/78 under theCMV promoter and the kinetics of rep expression is determined by WesternBlot with cell lysates collected at different time points aftertransfection. Next, these 293 cells are infected with Ad.AAV (MOI 1, 10,100 pfu/cell) at specific time points after transfection of Rep plasmidsdepending on the Rep expression kinetics: (e.g. 3, 6, 12, 24 . . . hoursafter transfection). It is important to time Ad.AAV infection exactlybecause viral DNA replication must be taking place or finished beforeRep production reaches peak levels. In general, adenovirus DNAreplication in 293 cells (infected with MOI 10) is maximal at 18 hourspost-infection, followed by production of structural proteins, packagingof viral genomes, and breakdown of cellular membrane structures (whichis concluded ˜36–48 h p.i.) (Shenk, T., 1996, In B. N. Fields, et al.(eds.), Fields Virology, vol. 2 Lippincott-Raven Publisher,Philadelphia; van der Vliet, B., 1995, In w. Doerfler, et al. (eds.)vol. 2 p. 1–31, Springer-Verlag, Berlin). Viruses are collected 48 hafter infection and banded by CsCl ultracentrifugation. Viral materialfrom purified bands corresponding to ΔAd.AAV is lysed, DNAse-treated (toliberate DNA associate Rep) and subjected to immunoprecipitation-WesternBlot with Rep specific antibodies to detect co-packaged Rep. Based ontheoretical calculations assuming that two Rep molecules bind per Adgenome, ˜1–10 np Rep proteins is expected from Lysates of 10¹⁰particles, which is within the range of detectability by Western Blot.Alternatively, co-packaged Rep may be detected based on its functionalactivity to mediate rescue and site-specific integration of the AAVITRtransgene cassette. To test whether functional Rep protein isco-packaged into hybrid vector particles, CsCl purified ΔAd.AAV1particles generated in 293 cells co-expressing Rep after Ad/AAV1infection (ΔAd.AAV1+Rep) can be used for transduction studies. Threedays after ΔAd.AAV1+Rep infection of the human cell line K562, cellularDNA is analyzed for AAVS1-specific integration events by PCR and PFGE.If efficient Rep-mediated site-specific integration of excised AAVcassettes is successful, then other ΔAd.AAV+Rep hybrid vectors withβ-Gal and SNori as transgenes can be produced.

Integration Studies with Rep Vectors in Erythroid Cells:

The hypotheses behind the rational of a rep-expressing hybrid vector(ΔAd.AAV-rep) are: (1) transient Rep co-expression from ΔAd.AAV-repvectors can enhance site-specific vector integration in human cells and(2) integration occurs via the AAV ITR(s) without the rep gene, which isplaced outside the AAV cassette, thus eliminating rep expression uponvector integration. To test hypothesis 1, transduction frequencies ofΔAd.AAV/rep versus ΔAd.AAV vectors can be compared based on theformation of G418 resistant colonies and quantify site-specificintegration events at different time points after infection of human andmouse cells by PFGE and PCR. To test hypothesis 2, the structure ofintegrated vector in transduced cell populations and single clones canbe delineated by Southern analysis and by sequencing ofvector/chromosomal DNA junctions. These studies can be performed withΔAd.AAV-rep, ΔAd.AAV, and ΔAd.AAV+Rep (copackaged protein) in human K562or HEL (for AAVS1 integration) and mouse MEL cell lines.

Cells infected with ΔAd.AAV-SNori, ΔAd.AAV-SNori+Rep orΔAd.AAV-Snori-rep can be subjected to G418 selection. The number of G418resistant colonies determined after 4 weeks of selection in relation tothe number of initially infected cells. The selection process forcolonies that did not survive continued selection due to potentialrep-mediated cytotoxcicity or episomal vector expression can bemonitored. If rep expression from ΔAd.AAV-SNori rep does not affect cellviability and proliferation, then more G418 resistant colonies shouldappear in ΔAd.AAV-SNori-rep and ΔAd.AAV-Snori+Rep. The structure ofintegrated vector can be determined by Southern Blot and sequencing ofintegration junctions.

To uncover a potential selection bias against rep producing cells aftertransduction with ΔAd.AAV/rep, site-specific and random vectorintegration events can be quantitated in cellular DNA isolated from cellpopulations at different time points after infection (e.g. 0.5, 1, 3, 7,14 days). To do this, the techniques based on PFGE-Southern can beutilized. It is expected that the signal(s) for AAVS1-specificintegration in ΔAd.AAV/rep infected human cells increases during thefirst days after infection and then remains constant over time.

In a separate study, the integration status of vector DNA (analyzed byPFGE or PCR) and the number of integrated copies (analyzed by SouthernBlot) with the expression level of β-galactosidase in single clonestransduced with β-Gal hybrid vectors (ΔAd.AAV-BG, ΔAd.AAV-BG+Rep, orΔAd.AAV-BG-rep) can be correlated. Together with data obtained in thestudies described in the Specification, this allows assessment ofwhether transcriptional silencing is associated with site-specificvector integration into the AAVS1 site.

It is not clear a priori whether the specific Rep function for vectorrescue, concatemerization, and integration can efficiently occur innon-S-phase or non-dividing cells. To test whether ΔAd.AAV^(fx), ΔAd.AAVor ΔAd.AAV-rep/+Rep vectors can integrate into non-dividing cells,transduction studies in cell cycle arrested cell cultures can beperformed as described earlier.

Discussion

The establishment of stable cell lines expressing Rep 68/78 atdetectable levels is not possible, which is probably due to rep mediatedcytotoxicity. Therefore, it is not possible to perform long-termtransduction studies (e.g. G418 selection or studies in single clones)in combination with ectopic rep expression. Moreover, due to theinhibitory effect of rep on adenovirus replication, it is currently notpossible to generate adenoviral vectors expressing rep under the RSV orPGK promoter.

Taken together, this indicates that co-expressed Rep may stimulatesite-specific transgene integration.

E. A Detailed Study of Transduction/Integration of Hybrid Vectors inErythroid Cell Lines:

In order to improve transduction and integration frequencies of thehybrid vectors into erythroid cell lines, a detailed study comparingvarious hybrid vectors have to be carried out as described below. Thetransduction studies are performed in K562 cells which is considered tobe an adequate model to study gene transfer vehicles into erythroidcells (Floch, V., et al., 1997, Blood Cells, Mol. and Diseases. 23,69–87). The optimal vectors should be able to integrate into thecellular genome with a high frequency, determined by Pulse field gelelectrophoresis (PFGE) and Southern blot as described in Example 4. Inaddition, the results from the following studies will serve to evaluatewhether a given hybrid vector needs to be modified for site-specificintegration in the host genome.

Sequencing of Integration Junctions:

The ultimate proof for vector integration is the sequencing of junctionsbetween SNori vector DNA and chromosomal DNA. Furthermore, thisclarifies the question whether the AAV ITRs represent the substrate forintegration. Specifically, DNA from clones with known ΔAd.AAVSNoriintegration structure (analyzed by Southern Blot) digested with EcoRI,which does not cut within the SNori cassette. The resulting fragmentsare circularized and transformed into a specific E. coli strain(according to the protocol described by Rutledge and Russell (Rutledge,E. A. et al., 1997, Journal of Virology, 71:8429–36)). Kanamycinresistant bacterial clones should contain the integrated SNori cassette.Flanking chromosomal DNA in rescued plasmids can be sequenced withprimers specific to the transgene.

To confirm vector integration in a small number of transduced cells,genomic DNA is extracted and digested with EcoRI. EcoRI fragments areligated to linkers containing a specific primer binding site and arethen digested with NotI, religated and propagated in E. coli. PlasmidDNA from a representative number of bacterial clones is sequenced todetermine the vector/chromosomal DNA junctions.

Dose Dependent Toxicity:

In order to test that the transduction frequence is dose-dependent andΔAd.AAV vectors, which are devoid of all adenoviral genes, could be usedto infect cells at higher doses with less cytotoxicity than firstgeneration adenovirus, K562 cells are infected with different MOIs(1–10⁸) of ΔAd.AAVBG and the first generation vector Ad.AAVBG (whichcontains the same β-Gal expression cassette). At day 4 post-infection,the total number of cells, the percentage of viable cells (based ontrypan blue exclusion) and the percentage of X-Gal positive cells arecounted. A fraction of infected cells are quantified for β-Galexpression using the Galacto-Light kit. The level of transgeneexpression is expected to be comparable between the two vectors. K562cells are predicted to tolerate higher doses of ΔAd.AAVBG better thanAd.AAVBG which express viral genes.

Integration Frequency with and Without G418 Selection:

In order to investigate the integration frequency of the differentvectors and to confirm that AAV ITRs present in double-strandedadenoviral DNA genomes can mediate vector integration with a frequencycomparable to rAAV vectors, integration studies are performed based onthe formation of G418 resistant colonies with ΔAd.AAVSNori, AdSNori,Ad.SNoriITR, and rAAVSNori after infection with 2×10⁵ and 2×10⁶ genomesper cell (FIG. 8). After infection, cells are plated in 96 well platesunder limiting dilution and selected with G418 to estimate the frequencyof formation of G418 resistant colonies. Another set of cells is platedwithout G418. A representative number of clones (w/ and w/o G418selection) are expanded to >10⁶ cells (after 3–4 weeks of culture) andanalyzed for the presence of viral DNA by Southern Blot as well as PFGEanalysis to discriminate between episomal vector DNA and vector genomesstably associated with chromosomal DNA. This allows us to estimate theintegration frequency of the different vectors, to assess the effect ofG418 selection on integration, and to consider position effects on neoexpression in calculating the total integration frequency. Integratedvector copies with a frequency of at least 1×10⁻⁴ is predicted only forΔAd.AAVSNori and rAAVSNori. The total number of colonies may be lower inboth the first generation vectors, Ad.AAVSNoriITR and Ad.SNori, due tothe toxic effects of expressed adenoviral proteins; however, a higherintegration frequency is predicted for the vector containing the AAVITRs (Ad.AAVSNoriITR).

Kinetics of Integration:

Compared to rAAV, the double-stranded nature of entering ΔAd.AAV genomesprovides more protection against degradation. Furthermore, the synthesisof transcriptionally active double-stranded intermediates fromsingle-stranded genomes, which is considered a limiting step in rAAVtransduction, is not required in ΔAd.AAV transduction. Thus, the lagphase between infection and expression seen with rAAV vectors, which iscausally linked to double-strand synthesis/integration may be shorter orabsent in infections with ΔAd.AAV vectors. Furthermore, it wasdemonstrated earlier that a 9 kb mini-adenoviral genome packaged intoadenoviral particles is only short lived and completely degraded by day3 post-infusion. In contrast, transduction with the 5.5 kb ΔAd.AAV1(FIG. 1) genome allows for long-term expression, suggesting that eitherAAV ITRs can stabilize the viral genome as an episome until it isintegrated or integration occurs shortly after infection.

The status of vector DNA can be examined in K562 cells at different timepoints after infection with ΔAd.AAVSNori, AdSNori, Ad.AAVSNoriITR, orrAAVSNori (MOI 2×10⁵). Infected cells are harvested at 1 hour, 5 hours,1 day, 3, 7, and 14 days after infection and chromosomal DNA is analyzedby PFGE followed by hybridization with a transgene specific probe. Thistechnique allows us to distinguish between episomal vector DNA, whichappears as a distinct 5.0 kb band and integrated DNA. Furthermore, extrachromosomal high-molecular weight vector concatemers can be detected. Inthe case of random integration, after digestion of chromosomal DNA withI-CeuI or PI-SceI, vector-specific signals in the range of 1–2 mb shouldbe seen. The intensity of episomal and integrated vector signal isquantified for each time point using phosphoimager analysis. This givesinformation about the kinetics of hybrid vector integration in apopulation of infected K562 cells and the intracellular stability ofhybrid vector genomes.

Structure of Integrated Vector DNA and Integration Functions withChromosomal DNA:

ΔAd.AAV1 integrates as concatemer/s randomly into host DNA as shownpreviously. How many vector copies are present in one concatemer andwhether the extent and the kinetics of tandem-formation are dosedependent still remain unclear. Another unanswered question is howΔAd.AAV integrates: whether one or both ITRs are involved, whether theintegrated ITRs are still intact, and whether adenoviral sequencesintegrate as well. These issues are important for the strategy toinclude rep genes into the hybrid vector genome. Moreover, if intact AAVITRs are present within integrated vector copies, helper virus(adenovirus or HSV) infection in vivomay mobilize the integrated AAV-ITRvector cassette and affect stability of transgene expression.

To answer these questions, K562 cells can be infected with ΔAd.AAVSNori,AdSNori, Ad.AAVSNorilTR, and rAAVSNori at MOIs 2×10⁵, 2×10⁶, or 2×10⁷genomes per cell. Infected cells are plated in 96 well plates in thepresence or the absence of G418. The latter is included because G418 maycause amplification of integrated vector DNA (Rutledge, E. A. et al.,1997, Journal of Virology, 71:8429–36). Genomic DNA from isolated clonescan be analyzed by regular Southern Blot as described in the ExamplesSection to confirm the presence of vector concatemers and calculate thenumber of integrated vector copies. More informative is the sequencingof integrated vector copies and their junctions with chromosomal DNA.The structure of integration junctions can be delineated using the roleof AAV ITRs in vector integration and the extent of insertionalmutagenesis after transduction. This data provides information about thepotential risks of hybrid vector used in clinical trials.

Transduction of Cell Cycle Arrested Cells:

The ultimate target for the hybrid vectors described in theSpecification are quiescent hematopoietic stem cells. We hypothesizethat the double-stranded nature of ΔAd.AAV genomes and specific nuclearimport mechanisms may allow for the transduction of non-dividing cells.This is in part supported by the transduction studies with ΔAd.AAV1 inquiescent hepatocytes in vivo. To confirm this data, primary fibroblastscan be forced to enter the G₀ phase by serum/growth factor starvationbefore infection with the hybrid vectors according to a protocoldescribed by Russell (Russell, D. et al., 1995, PNAS, 92:5719–23). Cellsare maintained for three days after infection under serum/growth factordeprivation. At this time point, genomic DNA is isolated and analyzedfor integration events by PFGE in comparison with growing cells. Anotherseries of integration studies can be carried out on K562 cells arrestedin the G/S phase of the cell cycle with aphidicolin (added 1 day beforeand maintained several days after infection with hybridvectors—depending on the integration kinetics studies describedearlier). To investigate whether DNA damaging agents increase thetransduction frequency of hybrid vectors, cell-cycle-arrested K562 cellsor primary fibroblasts can be treated with cisplatinum or ³H-thymidineprior to virus infection according to a protocol described by Alexanderand Russell (Alexander, I. E. et al., 1994, J. Virol., 68:8282–87;Russell, D. et al., 1995, PNAS, 92:5719–23). Furthermore, the effect ofchromosomal DNA decondensation on the transduction efficiency of hybridvectors can be studied in arrested cells after treatment with puromycin,staurosporin, Hoechst 3328, distramycin, or vandate.

F. Improvements in ΔAd.AAV Production and Purification

To inhibit packaging of full-length genomes a modified form of I-Sce I,a yeast mitochondrial intron-endonuclease with a non-palindromic 18-bprecognition sequence is expressed in 293 cells. Constitutive expressionof this enzyme in mammalian cells is not toxic, possibly due to eitherthe lack of I-SceI sites in the genome or sufficient repair of them(Rouet P. et al, 1994, PNAS, 91:6064–8). The yeast I-Sce I is modifiedwith an SV40 T-antigen nuclear localization signal and an optimal Kozaksequence to enhance its functionality in mammalian cells (Rouet P. etal, 1994, PNAS, 91:6064–8). For another yeast endonuclease it was shownthat a recognition site within an transduced Ad genome was efficiently(30% of all transduced genomes) when expressed in human A549 cells.Importantly, the expression of E4 ORF6 and ORF3 expressed from thetransduced Ad genome inhibited double-strand break repair mediated bythe endonuclease (Nicolas, A. L. et al, 2000, Virology, 266:211–24).This is consistent with the observations by others where these E4proteins prevent concatemerization of the viral genome (Boyer, J. et al,1999, Virology, 263:307–12). Based on this, packaging of full-lengthvirus containing a I-Sce1 recognition site is reduced in 293 cellsconstitutively expressing I-Sce I. The 18mer I-Sce site is inserted intothe E3 region of the Ad.IR vectors. These vectors are generated andamplified in 293 cells followed by a large-scale infection of 293 cellsexpressing I-SceI. Alternatively, an expression cassette for theendonuclease Xhol is inserted into the E3 region of Ad.IR or Ad.AAVvectors. The Xhol, gene will be modified for optimal function inmammalian cells. Vectors expressing Xhol are generated and amplified in293 cells expressing the Xho I isoschizomer PaeR 7 methyltransferase(PMT) (Nelson, J. E. et al, 1997, J. Virol., 71:8902–7), which mediatesthe addition of a methyl group onto the N6 position of the adenine baseof Xho I sites, CTCGAG. This protects the viral and cellular genome fromXhol cleavage. Methylated Ad vectors are produced at high titers.ΔAd.AAV vectors are then obtained by large-scale infection of 293 cellswith the Ad.AAV-Xhol vectors. At this stage the viral genome is notmethylated and is digested at the Xhol sites. Xhol sites present withinthe transgene cassette are deleted by site-directed mutagenesis withoutaltering the amino acids sequence. (Xhol is accumulated only at latestages in virus replication and should act only upon a large part of AdDNA when replication is completed. In addition, ultracentrifugationoptimizes the separation between ΔAd.IR and ΔAd.IR particles (Blague, C.et al., 2000, Blood, 95:820–8).

EXAMPLE II

Modified Fiber Protein

A. Test the Infectivity of Different Human or Animal Serotype on HumanBone Marrow Cells.

Since the amino acid sequence of the fiber knob region variesconsiderably among the ˜50 known serotypes, it is thought that differentadenovirus serotypes bind to different cellular receptor proteins or usedifferent entry mechanisms (Shenk, T., 1996, In B. N. Fields, et al.(eds.), Fields Virology, vol. 2 Lippincott-Raven Publisher,Philadelphia; Mathias, P. et al., 1994, Journal of Virology, 68:6811–14;Defer, M., et al., 1993, J. of Virology, 64, 3661–3673). Although mostadenoviruses contain RGD motifs in the penton base proteins, there are anumber of serotypes (e.g. Ad 40, 41) without this conserved sequence.These types may use integrin □v-independent pathways for virusinternalization (Davison, A. J., et al., 1993, J. Mol. Biol., 234,1308–1316; Mathias, P. et al., 1994, Journal of Virology, 68:6811–14).To test whether other Ad serotypes can infect stem cell subpopulationpresent in human bone marrow, studies with a series of different humanAd serotypes and animal viruses can be performed (see Table II). As ameans to verify efficient transduction with Ad serotypes, viral DNA istagged before infection and the presence of viral genomes in the nucleiof transduced cells is investigated. Furthermore, whether viral DNA isreplicated in transduced cells can be analyzed as indirect proof forearly viral gene expression. A direct detection of expressed viralproteins is impossible due to the unavailability of antibodies againstall the serotypes included in this study. Simultaneously with theinfection assay, transduced human bone marrow cells can be analyzed formorphological and immunohistochemical features characteristic of HSC orprogenitor subpopulations. For retargeting, serotypes which are able toinfect CD34+subsets of bone marrow cells at the lowest MOI are selected.As the next step, the fiber gene is PCR-cloned from serotypes withpotential HSC/CD34+tropism and inserted into standard shuttle plasmidsfor Ad5 vector generation replacing the Ad5 fiber gene using an E. colirecombination system (FIG. 9).

Methods

Cells and Viruses:

HeLa (human cervix carcinoma, ATCC CCL-2.2), CHO (chinese hamster ovary,ATCC CCL-61), K562 (human hematopoietic, ATCC 45506), HEp-2 (humanlarynx carcinoma, ATCC CCL-23), 293 (human embryonic kidney, Microbix,Toronto Canada) cells were maintained in DMEM, 10% FCS, 2 mM glutamine,and Pen/Strep. Culture media for CHO cells was supplemented with 200 μMasparagine and 200 μM proline. Human CD34+-enriched bone marrow cellswere purified from peripheral blood after mobilization using MiniMACSVS⁺ separation columns (Miltenyi Biotec, Auburn, Calif.), according tothe manufacturer's instructions. Aliquots were stored in liquidnitrogen. Sixteen hours before the experiment, cells were recovered fromthe frozen stock and incubated overnight in IMDM media, supplementedwith 20% FCS, 10⁻⁴ M β-mercaptoethanol, 100 μg/ml DNaseI, 2 mMglutamine, 10 U/ml IL-3, and 50 ng/ml stem cell factor (SCF) or 2 ng/mlthrombopoietin (Tpo). The purity of CD34+preparations was verified byflow cytometry and was consistently greater than 90%.

Flow Cytometry:

Adherent cells (CHO, HeLa) grown in non-tissue culture treated 10 cmdishes (Falcon, Franklin Lakes, N.J.) were detached by treatment with 1mM EDTA and washed three times with wash buffer (WB), consisting of PBSsupplemented with 1% FCS. Cells grown in suspension (K562, CD34+) werewashed three times with WB. After washing, cells were resuspended in WBat 2×10⁶ cells/ml. 2×10⁵ cells were incubated in WB for 1 h at 37° C.with monoclonal antibodies specific for α_(v)-integrins [L230, ATCC:HB-8448, (Rodriguez, E., Everitt, E. 1999. Arch. Virol. 144:787–795)(1/30 final dilution), CAR [RmcB (Bergelson, J. M., et al. 1997.Science. 275:1320–1323; Hsu, K.-H., L., et al. 1988. J Virology.62:1647–1652) (1/400 final dilution)], or BrdU [(Amersham, ArlingtonHeights, Ill.) (1/100 final dilution)]. Subsequently, cells were washedwith WB, and incubated with fluorescein isothiocyanate (FITC)-labeledhorse anti-mouse IgG antibodies [(Vector Labs., Burlingame, Calif.)(1/100 final dilution)] or phycoerythrin (PE)-labeled goat anti-mouseIgG antibodies [(Calbiochem, La Jolla, Calif.) 1:100 dilution] for 30min at 4° C. After incubation with secondary antibodies, cells werewashed two times with WB and 10⁴ cells per sample were analyzed induplicate by flow cytometry.

For the analysis of CD34 and c-kit expression on transduced CD34+-cellsand for fluorescent activated cell sorting (FACS), purified human CD34+cells were incubated with phycoerythrin(PE)-conjugated anti-CD34monoclonal antibodies (Becton-Dickinson Immunocytochemistry Systems, SanJose, Calif.) or with PE-labeled anti-CD117 (c-kit) monoclonalantibodies (MAb 95C3, Immunotech, Beckman Coulter, Marseille, France)according to the manufacturer's protocol followed by flow cytometryanalysis. All analyses and sortings were performed on a FACStar Plusflow cytometer (Becton Dickinson, Franklin Lakes, N.J.) equipped with488 nm argon and 633 nm HeNe lasers. For analysis of c-kit expressionand FACS purification of CD34+/c-kit+ cells, SCF was not added to themedia during culturing of CD34+ cells.

Results

CAR/α_(v)-Integrin Expression on Test Cells:

It is generally accepted that CD34+ cells possess bone marrowrepopulating activity. Therefore, we used human CD34+ cells as thetarget for our studies towards identifying Ad serotypes with HSC tropismand constructing new viral vectors. Studies were performed on mobilized,CD34-positive, peripheral blood cells from one donor under conditionswhich are known to retain CD34+ cells in a quiescent stage (Leitner, A.,et al. 1996. Br. J. Haematol. 92:255–262; Roberts, A. W., Metcalf, D.1995. Blood. 86:1600–1605). More than 90% of purified cells were CD34positive by flow cytometry. Furthermore, we included into our Ad tropismstudies the cell line K562, which is considered to be an adequate modelfor studying gene transfer into human hematopoietic cells (McGuckin, etal. 1996. British Journal of Haematology. 95:457–460). HeLa cells, whichare readily infectible by Ad5, and CHO cells, which are refractory toAd5 infection (Antoniou, M. et al., 1998, Nucleic Acid Res., 26:721–9),were used as positive and negative control cell lines, respectively.

For Ad5, both, binding to the primary receptor and to α₃β₅ and α_(ω)β₅integrins are important for high efficiency infection of target cells.The expression of CAR and α_(v) integrins on test cells was analyzed byflow cytometry using monoclonal antibodies against CAR (RmcB (Bergelson,J. M., et al. 1997. Science. 275:1320–1323; Hsu, K.-H., L., et al. 1988.J Virology. 62:1647–1652)) and α_(v) integrins (L230 (Roelvink, P. W.,et al. 1996. J Virology. 70:7614–7621)) (FIG. 10). As expected, nearlyall HeLa cells expressed high levels of CAR and α_(v)-integrins, whereasCHO cells lacked significant CAR and α_(v)-integrin expression. Fifteenand 77% of K562 cells expressed CAR and α_(v)-integrins, respectively.Only ˜6% of the CD34+ cells used in our studies expressed CAR and 17%were positive for α_(v)-integrins. Notably, the preparation of CD34+cells represents a mixture of different cell types. The absent or lowexpression of primary and secondary Ad5 receptors on non-cycling humanCD34+ cells is in agreement with previous reports (Huang, S., et al.1996. J Virology. 70:4502–4508; Neering, S. J., et al. 1996. Blood.88:1147–1155; Tomko, R. P., et al. 1997. Proc. Natl. Acad. Sci. USA.94:3352–3356).

Infection Assay Using Wild-Type Ad5 and K562 Cells:

The presence of viral DNA in the nucleus of infected cells is anindirect means to demonstrate efficient virus binding, internalization,and nuclear import. Nuclear localization of the viral genome is aprerequisite for transgene transcription and integration. Two techniquesare utilized to tag viral DNA for in situ analysis. To optimize theinfection assay, wild-type Ad5 virus and K562 cells which are permissivefor Ad5 infection can be used. The first protocol (Challberg, S. S. andKetner, S. 1981, Virology 114, 196–209), is based on ³²P-labeling ofviral DNA. During amplification of wild-type Ad5 and A549 cells,³²P-phosphate (40 μCi/ml) is added to phosphate-free medium. Afterdevelopment of CPE, ³²P-tagged virus is harvested, banded in CsClgradients, and titered on HeLa cells according to standard protocols. Tosimulate the conditions for infection of human bone marrow cells, K562cells are incubated in suspension with a MOI of 1, 10, or 100 of ³²P-Ad5for 2, 4, 6, or 8 hours under agitation at 37° C. This covers the timeperiod necessary for adsorption, internalization, and nuclear import.After washing, cells are fixed either transferred to microscopy slidesusing cytospin or embedded in paraffin and sectioned (according toprotocols from VECTOR labs, Burlingham, Calif.). The latter has thepotential advantage that multiple consecutive sections (5 μm) of thesame cell can be analyzed by different methods (e.g. for ³²P taggedviral DNA, for specific histological staining, for immunofluorescence),which allows for correlating infection with a particular cell typepresent in the bone marrow. Cells are incubated in a Kodak NTB-2 photoemulsion for autoradiography. The exposure time can be optimized tominimize background or non-nuclear localized signals. A dose and timedependent appearance of nuclear silver grains is expected under theoptimized conditions. Since ³²P-phosphate can label viral proteins aswell, a cytoplasmic background signal might appear. To facilitatedetection, immunofluorescence with HSC specific antibodies on sectionscan be performed. As an alternative method, a BrdU-labeling techniquefor viral DNA can be used (Lieber, A., et al. 1999. J Virol 73:9314–24;Lieber, A. et al., 1996, Journal of Virology, 70:8944–60). In this case,different amounts of BrdU are added to the A549 culture medium duringwtAd5 virus propagation. BrdU labeled viral DNA can be detected withmonoclonal antibodies specific to BrdU. The signal can be enhanced usinglayers of species-specific polyclonal antibodies in combination withbiotin/avidin and a fluorescent marker. BrdU tagged viral DNA can bedetected on cytospins of bone marrow cells together with cell surfacemarkers by double or triple immunoflourescence.

Discussion

The interaction of selected Ad serotypes with CD34+ cells was tested. Asa result of this screening we constructed a first-generation, Ad5-basedvector whose fiber was substituted with the fiber derived from Ad35. Wedemonstrated that this capsid modification allowed for efficient viraltransduction of potential HSCs by the corresponding chimeric Ad vectors.

All tropism and transduction studies were performed with non-cyclingCD34+ cells, which are thought to include HSCs. The quiescent stage ofCD34+ cells purified from mobilized blood is important because inductionof cell proliferation is associated with a loss of the ability toreconstitute hematopoiesis and with changes in the spectrum of cellularreceptors (Becker, P. S., et al. 1999. Exp. Hematol. 27:533–541). It isknown that treatment of hematopoietic cells with cytokines or growthfactors changes the expression of specific integrins includingα_(v)-integrins, which would ultimately alter the susceptibility ofcells to Ad infection or may effect viability of infected cells(Gonzalez, R., et al. 1999. Gene Therapy. 6:314–320; Huang, S., et al.1995. J. Virology. 69:2257–2263). Another fact that complicates theinterpretation of transduction studies is the extraordinaryheterogeneity of CD34+ cells in regards to morphology and function.

B. Screening Different Adenoviruses to Establish Tropism to HSC.

The ATCC provides more than 70 different human or animal adenoviruses(see Appendix I). A collection of 15 human serotypes and 6 animaladenoviruses (see Table II) are selected based on the followingcriteria: (i) availability of the complete genome sequence or fibersequence from the NIH gene bank (ii) CAR receptor usage absent orunknown, (iii) different subgroups, and (iv) moderate or lowtumorigenicity (Shenk, T., 1996, In B. N. Fields, et al. (eds.), FieldsVirology, vol. 2 Lippincott-Raven Publisher, Philadelphia). However, anyserotype shown in the Appendix hereto can be used for the inventiondescribed. Animal viruses are included in the infectivity assay becausethis may provide a means to circumvent the pre-existing humoral immunityagainst human Ad5 fiber, which represents a critical obstacle forclinical trials with Ad vectors.

Methods

Viruses:

The following human adenovirus serotypes were purchased from the ATCC: 3(VR-3), 4 (VR1081), 5 (VR-5), 9 (VR1086), 35 (VR-716) and 41 (VR-930).Adenovirus No. VR-716 was purchased from ATCC labeled as serotype 34,however it was found to be serotype 35 upon sequencing of the fiberregion. For amplification, the corresponding Ads were infected ontoHeLa, 293, or HEp-2 cells under conditions that preventedcross-contamination. Virus was banded in CsCl gradients, dialyzed andstored in aliquots as described elsewhere (Lieber, A., C.-Y. et al.1996. Journal of Virology. 70:8944–8960). Plaque titering was performedas follows: Confluent 293 cells plated in 6-well plates were incubatedfor 24 hours with virus in a total volume of 1 ml. Two weeks afterinfection, plaques were counted on cultures overlayed with 1%agarose/MEM/10% FCS.0

EM Studies:

CsCl-banded Ad stocks were thawed and diluted with 0.5% glutaraldehyde.Grids were prepared as described earlier (Mittereder, N., et al. 1996.J. Virology. 70:7498–7509). After staining with 2% methylamine tungstate(Nanoprobes, Stony Brook, N.Y.), the carbon-coated grids were evaluatedand photomicrographed with a Phillips 410 electron microscope, operatedat 80 kV (final magnification 85,000×). For each particular Ad serotype,the number of morphologically deficient viral particles per 100 wascounted in five random fields.

Results

Electron Microscopy:

Little is known about the stability of particles from serotypes otherthan Ad5. Since the intactness of viral particles was crucial forcomparative interaction studies, virions from the serotypes specifiedabove were analyzed by electron microscopy (EM). EM studies of negativecontrast stained Ad suspensions demonstrated that the percentage ofdefective particles (loss of icosahedral shape or luminal staining) didnot exceed 5% indicating that serotype preparations had comparablequalities. Representative EM photographs are shown: for Ads 5, 9, and 35(FIG. 11).

Serotype Screening:

It is thought that different Ad serotypes bind to different cellularreceptor proteins and use different entry mechanisms (Defer, C., et al.,P. 1990. J. Virology. 64:3661–3673; Mathias, P., et al. 1994. Journal ofVirology. 68:6811–6814). A set of human adenoviruses was obtained fromthe ATCC to be tested for tropism to CD34+ cells. These includedserotypes 3, 4, 5, 9, 35, and 41 representing different subtypes (Table1). We believed that these serotypes would use different cellularattachment and internalization strategies due to differing lengths offiber shafts (Chroboczek, J., et al. 1995. Adenovirus fiber, p. 163–200.In a. P. B. W. Doerfler (ed.), The molecular repertoire of adenoviruses,vol. 1. Springer Verlag, Berlin; Roelvink, P. W., et al. 1998. J.Virology. 72:7909–7915), the presence or absence of RGD motifs withinthe penton base, and differing tissue tropism. The relatively littlecharacterized Ad35 was selected because it was found inimmunocompromised hosts, particularly in bone marrow recipients(Flomenberg, P., et al. 1994. Journal of Infectious Diseases.169:775–781; Flomenberg, P. R., et al. 1987. Journal of InfectiousDiseases. 155:1127–1134; Shields, A. F., et al. 1985 New England Journalof Medicine. 312:529–533). The latter observations prompted us tobelieve that bone marrow cells are among the natural reservoirs forAd35.

TABLE II Human and animal adenoviruses with potential interest for theinvention Human/ Human/ Human/ Adenovirus Group B Group D Group F AvianBovine Canine Ovine Swine Mouse Serotype 3, 7, 11, 16, 8, 15, 17, 40, 41CELO, 3 1, 2 5 4 1 21, 34, 35 19, 28, 37 EDS The underlined serotypesuse CAR independent pathways for cell entry.

For amplification, the corresponding adenovirus stocks can be infectedonto HeLa or A549 cells such that at a given time only one virus type ishandled in a separate laminar flow hood and cultured in Hepa-filteredbottles, preferentially in separate CO₂ incubators to avoidcross-contamination. During propagation, viral DNA is tagged using oneof the techniques described earlier. Viral DNA can be isolated frompurified particles. The XhoI restriction pattern is analyzed formethylated and unmethylated viral DNA by Southern blot using the fullgenome of the corresponding virus type as a radioactive probe.

Discussion

Although it was reported earlier by slot-blot assay that fiber knobsderived from 2, 9, 4, and 41 L can bind to CAR (Roelvink, P. W., et al.1998. J. Virology. 72:7909–7915), it is not clear whether this bindingoccurs with an affinity that is physiologically relevant and whetherthis would confer cell entry. Furthermore, as shown for the Ad5interaction between the penton and intergrins, a secondary receptor isrequired to induce virus internalization. We demonstrated that differentserotypes interacted differently with the K562 or CD34+target cells.Ad5, Ad4, and Ad41 were not able to efficiently attach to and beinternalized by K562 and CD34+ cells. Although Ad4 belongs to a separatesubgroup (E), it is thought that Ad4 represents a natural hybrid betweensubgroup B and C viruses with a fiber related to Ad5 (Gruber, W. C., etal. 1993. Virology. 196:603–611). Therefore, it was not surprising thatAd4 has binding properties similar to Ad5. The subgroup F serotype Ad41has been shown to contain distinct fibers, a long shafted and ashort-shafted fiber allowing for different cell entry pathways(Tiemessen, C. T., Kidd, A.H. 1995. J. Gen. Virol. 76:481–497). The Ad41penton base does not contain RGD motifs suggesting that this virus mayuse α_(v)-intregrin independent pathways for cell entry. However, thesefeatures did not improve interaction with CD34+ cells. Ad9, Ad3, andAd35 did interact with CD34+ cells more efficiently than Ad5. Out of allthe serotypes tested, Ad35 demonstrated the most efficient attachmentand internalization with K562 and CD34+ cells. Although theshort-shafted Ad9 can bind to CAR, it preferentially usesα_(v)-integrins for cell entry (Roelvink, P. W., et al. 1996. J.Virology. 70:7614–7621). Therefore, the low level of α_(v)-integrinexpression on certain subsets of CD34+ cells may account for theobserved susceptibility to Ad9.

C. Attachment and Internalization of the Ad Serotypes to K562 and CD34+Cells.

Methods

Labeling of Ads with [³H]-methyl thymidine:

Serotypes were labeled with [³H]-methyl thymidine as described in detailelsewhere (Roelvink, P. W., et al. 1996. J. Virology. 70:7614–7621).Briefly, 5×10⁷ HeLa or 293 cells were grown in 175 sq. cm flasks with 15ml DMEM/10% FCS and infected with wild type adenovirus at a MOI of 50 orhigher. Twelve hours post-infection, 1 mCi of [³H]-methyl thymidine(Amersham, Arlington Heights, Ill.) was added to the media and cellswere further incubated at 37° C. until complete CPE was observed. Then,cells were harvested, pelleted, washed once with cold PBS, andresuspended in 5 ml PBS. Virus was released from the cells by fourfreeze-thaw cycles. Cell debris was removed by centrifugation and viralmaterial was subjected to ultracentrifugation in CsCl gradients andsubsequent dialysis as previously described (Lieber, A., C.-Y. et al.1996. Journal of Virology. 70:8944–8960). Virus purification anddialysis removed unincorporated radioactivity. Wild type Ad particleconcentrations were determined spectrophotometrically by measuring theOD₂₆₀, utilizing the extinction coefficient for wild-type Ad5ε₂₆₀=9.09×10⁻¹³ OD ml cm virion⁻¹ (Maizel, J. V., et al. 1968. Virology.36:115–125). The virion specific radioactivity was measured by a liquidscintillation counter and was always in the range of 1×10⁻⁵ to 1×10⁻⁴cpm per virion. For selected variants, the fiber gene was PCR amplifiedand sequenced to ensure identity and the absence of cross-contamination.

Viral DNA Tagged with Methylase and Test for Replication by GenomicSouthern Blots:

To ultimately confirm transduction, a protocol to detect adenoviralreplication in infected cells can be established. Viral DNA synthesiscan only occur after de novo expression of adenoviral early genes. Asite-specific methylation strategy is utilized to monitor viral DNAreplication within infected cells (Nelson, J. et al., 1997, Journal ofVirology, 71:8902–07). Methylation marked adenovirus can be produced bythe addition of a methyl group onto the N6 position of the adenine baseof XhoI sites, CTCGAG, by propagation of the virus in HeLa or A549 cellsexpressing the XhoI isoschizomer PaeR7 methyltransferase (PMT) (Kwoh, T.J., et al., 1986, Proc. Natl. Acad. Sci. USA 83, 7713–7717). It is knownthat methylation does not affect vector production but does preventcleavage by XhoI. Loss of methylation through viral replication restoresXhoI cleavage and can be detected by Southern blots of genomic DNA frominfected cells in comparison to native, non-methylated, viral genomes.

Attachment and Internalization Assays:

These studies were performed based on a protocol published elsewhere(Wickham, T. J., et al. 1993. Cell. 73:309–319). In preliminaryexperiments, we found that labeled Ad5 virions reached equilibrium inattachment to HeLa cells after 45 min at 4° C. with an MOI of 400 pfuper cell. For attachment studies, 3.5×10⁵ cells were incubated for onehour on ice with equal amounts of [³H]-labeled adenovirus OD particlesequivalent to an MOI of 400 pfu/cell for Ad5 in 100 μl of ice-coldadhesion buffer (Dulbeco's modified Eagle's medium supplemented with 2mM MgCl₂, 1% BSA, and 20 mM HEPES). Next, the cells were pelleted bycentrifugation for 4 min at 1000×g and washed two times with 0.5 mlice-cold PBS. After the last wash, the cells were pelleted at 1500×g,the supernatant was removed, and the cell-associated radioactivity wasdetermined by a scintillation counter. The number of viral particlesbound per cell was calculated using the virion specific radioactivityand the number of cells. To determine the fraction of internalized[³H]-labeled adenoviral particles, cells were incubated on ice for onehour with the corresponding virus, washed with PBS as described above,resuspended in 100 μl adhesion buffer, and then incubated at 37° C. for30 min. Following this incubation, cells were diluted 3-fold with cold0.05% trypsin-0.5 mM EDTA solution and incubated at 37° C. for anadditional 5–10 min. This treatment removed 99% of attachedradioactivity. Finally, the cells were pelleted at 1500×g for 5 min, thesupernatant was removed, and the protease-resistant counts per minutewere measured. This protocol minimizes the possibility that theinternalization data were affected by receptor recycling (Rodriguez, E.,Everitt, E. 1999. Arch. Virol. 144:787–795). Nonspecific binding of Adparticles to cells on ice was determined in the presence of 100-foldexcess of unlabeled virus. This value routinely represented less than0.1% of viral load.

Results

Attachment of Ad Particles to Target Cells and Internalization:

The selected serotypes were metabolically labeled with [³H]-thymidine,which is incorporated into viral DNA during replication. Adsorption andinternalization can be experimentally dissociated by taking advantage ofthe observation that at low temperature (0–4° C.) only virus cellattachment occurs, whereas internalization requires incubation at highertemperatures. The number of particles adsorbed or internalized per cellwas calculated using the virion-specific radioactivity and used toquantify interaction of Ads 3, 4, 5, 9, 35, and 41 with CD34+, K562,HeLa and CHO cells (FIG. 12). The serotypes varied significantly intheir ability to attach to and to be internalized by the different celllines. For Ad5, the degree of attachment to the cell lines testedcorrelated with the level of CAR expression. In CHO cells, which werepreviously shown to be refractory to Ad5 infection, the level ofattachment and internalization was about 50–70 viral particles per cell.This number was hereafter assumed negative in terms of susceptibility ofa given cell type for Ad5. Interaction of the other serotypes with CHOcells was not significantly higher indicating that correspondingreceptor/s were absent on CHO cells. All serotypes tested interactedwith HeLa cells; with Ad3 and Ad35 being the most efficient variants.The presence of distinct Ad3 and Ad5 receptors on HeLa cells wasdemonstrated previously (Stevenson, S. C., et al. 1995. J. Virology.69:2850–2857). Ads 4, 5, and 41 did not bind to K562 cells. In contrast,Ad9 as well as the members of subgroup B, Ad3 and Ad35, efficientlyinteracted with K562 cells with Ad35 having the highest number ofadsorbed and internalized particles. Compared to Ad5, about 25 timesmore Ad35 particles were attached and three-forth of these wereinternalized by K562 cells. Viral interactions with CD34+ cells weregenerally weaker. Among the serotypes tested, only Ad9 and Ad35 weresignificantly internalized by non-cycling CD34+ cells. Internalizationof Ad9 and Ad35 was, respectively, four and eight times more efficientthan for Ad5 particles. The number of Ad35 virions internalized by CD34+cells was almost half of that seen for Ad5 in HeLa cells, which can bereadily infected with Ad5 based vectors.

Attachment and Internalization of Adenovirus Serotypes 3, 5, 9, 35 and41 into Hela, 293, and CHO Cells:

Hela and 293 cells expressing high level of primary and secondaryreceptors for human adenoviruses are used as a positive control forvirus attachment and internalization. As a negative control CHO cellsare used. CHO cells do not express the primary adenoviral receptor at adetectable level, and are therefore refractory for adenoviral infection.For attachment studies, these adherent cell lines are detached from 10cm dishes with PBS-EDTA solution (without Ca2+ and Mg2+), washed threetimes with ice-cold PBS, resuspended in adhesion buffer, and incubatedwith viruses as described above in the Examples section. As expected,all adenoviral serotypes tested are efficiently attached to andinternalized into Hela cells (Table III) (FIG. 13). Adenovirusesserotypes 3, 5, 35, 41, but not 9, are efficiently attached to andinternalized by 293 cells. In contrast, poor attachment andinternalization of most adenovirus serotypes are observed with CHOcells. The level of attachment on CHO is about 50–70 virus particles percell for adenoviruses serotypes 5 and 41, 115 virus particles per cellfor adenovirus type 3 and about 180 particles per cell for adenovirusserotypes 9 and 35. For further analysis, numbers >300 viral particlesper cell are assumed as positive and <70 viral particles per cell asnegative in terms of susceptibility of a particular cell line forefficient adenoviral transduction.

TABLE III Comparative analysis of attachment and internalization of Ad5and Ad9 to cell lines, expressing different amounts of CAR andαυβintegrins. Ad9 Ad5 CAR αυβ-integrin (attached/ (attached/ Cell lineexpression expression internalized) internalized) HeLa ++ ++ 426/370550/500 CHO − ++ 300/300 70/50 293 ++ ++ 20/20 1950/1750 Y79 +++ −190/140 1200/1100 K562 − + 320/230 60/50 Erythrocytes ? ? 420/—  68/—Attachment and Internalization of Adenovirus Serotypes 3, 5, 9, 35 and41 into Human CD34+ Bone Marrow Cells and K562 Erythroleukemia CellLine:

Previous studies showed that the human erythroleukemia cell line K562can be transduced with Ad5-based adenoviral vectors at very high MOIs.As shown in FIG. 14, only about 60 viral particles per cell ofadenovirus serotype 5 are attached to and even fewer particles areinternalized into these cells at a MOI of 400. In contrast to Ad5, about320 viral particles per cell of Ad9 and about 1500 viral particles percell of Ad35 are attached to and about two-thirds of them areinternalized into K562 cells (FIG. 14B). Human unstimulatedCD34+-enriched bone marrow cells obtained from frozen stocks areincubated overnight in growth medium without cytokine stimulation. Thenext day, the number of viable cells is calculated. For attachmentstudies, cells are washed three times with ice-cold PBS, resuspended inadhesion buffer and incubated with adenoviruses. Among the adenoviralserotypes tested, only adenovirus particle of Ad9 (about 150 viralparticles per cell) and Ad35 (about 320 viral particles per cell) areable to attach to unstimulated CD34+ cells on the level, compared to Ad5 (only 60 viral particles per cell). Four-fifths of these virusparticles are able to be internalized by the cells. Interestingly, uponstimulation of CD34+ cells with GM-CSF and EPO/TPO for two weeks,attachment and internalization of Ad9 viral particles are significantlyincreased (up to 300 particles per cell). At the same time, thetransient stimulation of cells with GM-CSF for two days could notincrease the level of viral attachment to the cells.

Based on the above finding that Ad35 serotype is able to attach andinternalize into CD34+ cells most efficiently among several serotypestested, serotype Ad35 was selected for further studies. As described inAppendix II, a chimeric vector (Ad5 GFP/F35) containing theshort-shafted Ad35 fiber sequence in an Ad5 capsid was able to target abroad spectrum of CD34+ cells in a CAR/integrin independent manner.

Discussion

In summary, from all the serotypes tested, Ad9, Ad3, and Ad35demonstrated the most efficient attachment to and internalization withK562 and CD34+ cells. Based on adsorption/internalization data, Ad9 andAd35 as representatives for subgroups D and B were selected for furthertropism studies.

D. Characterization of Ad Vector Replication in K562 and CD34+ Cells.

Comparative analysis of Ad5, and Ad9 and Ad34 to infect and to replicatein 293, K562 and CD34+ cells. The ability of the Ad9 fiber knob domainto recognize the same primary receptor on the cell surface as Ad5 withcomparable affinity was described earlier. Thus, the finding that Ad9viral particles can only poorly attach to 293 cells is ratherunexpected. In order to find out how the attachment and internalizationdata reflect the biological activity of adenoviruses of differentserotypes, the stocks of Ad5, Ad9 and Ad35 are characterized in moredetail by electron microscopy, plaque assay on 293 cells, andquantitative replication assay in K562 and CD34+ cells.

Methods

Quantitative Replication Assay:

1×10⁵ CD34+ or K562 cells were infected in 100 μl of growth media withdifferent MOIs of Ad5, 9, or 35 which had been amplified in 293 cells,expressing the XhoI DNA methyltransferase isoshizomer PaeR7 (Nelson, J.,Kay, M. A. 1997. Journal of Virology. 71:8902–8907). After 2 hours ofincubation at 37° C., the cells were centrifuged at 1000×g for 5 min,the virus-containing medium was removed, the cells were resuspended in100 μl of fresh media, and then they were incubated at 37° C. untilharvesting. At 16 hours post-infection for K562 cells, or 36 hpost-infection for CD34+ cells, 5 μg of pBS (Stratagene, La Jolla,Calif.) plasmid DNA was added as a carrier which could also be used as aloading control. Genomic DNA was extracted as described previously(Lieber, A., C.-Y. et al. 1996. Journal of Virology. 70:8944–8960).One-fourth of purified cellular DNA (equivalent to 2.5×10⁴ cells) wasdigested with HindIII, XhoI, or with HindIII and XhoI together at 37° C.overnight and subsequently separated in a 1% agarose gel followed bySouthern blot with chimeric Ad5/9 or Ad5/35 DNA probes. The chimericprobes, containing sequences of Ad5 and Ad9 (Ad 5/9) or Ad5 and Ad35 (Ad5/35), were generated by a two-step PCR amplification using Pfu-TurboDNA polymerase (Stratagene, La Jolla, Calif.) and viral DNA frompurified particles as a template. The following primers were used forPCR (Ad5 sequences and nucleotide numbers are underlined): Ad5F1-(nt:32775–32805) 5′-GCC CAA GAA TAA AGA ATC GTT TGT GTT ATG-3′ (SEQ ID NO.:3): Ad5R1-(nt: 33651–33621) 5′-AGC TGG TCT AGA ATG GTG GTG GAT GGC GCCA-3′ (SEQ ID NO.:4): chimeric Ad5/9F-(nt: 31150–31177, nt: 181–208)5′-AAT GGG TTT CAA GAG AGT CCC CCT GGA GTC CTG TCA CTC AAA CTA GCT GACCCA-3′ (SEQ ID NO.: 5): chimeric Ad5/9R-(nt: 32805–32775, nt:1149–1113)5′-CAT AAC ACA AAC GAT TCT TTA TTC TTG GGC TTC ATT CTT GGG CGA TAT AGGAAA AGG-3′ (SEQ ID NO.:6); chimeric Ad5/35F-(nt: 31150–31177, nt:132–159) 5′-AAT GGG TTT CAA GAG AGT CCC CCT GGA GTT CTT ACT TTA AAA TGTTTA ACC CCA-3′ (SEQ ID NO.:7), chimeric Ad5/35R (nt: 32805–32775, nt:991–958) 5′-CAT AAC ACA AAC GAT TCT TTA TTC TTG GGC ATT TTA GTT GTC GTCTTC TGT AAT GTA AG-3′ (SEQ ID NO.:8). Nucleotide numbers are givenaccording to the sequences obtained from the NCBI GenBank (accession No.M73260/M29978 for Ad5, X74659 for Ad9, and U10272 for Ad35). After thefirst amplification, the 968 bp-long Ad9, a 859 bp-long Ad35 DNAfragments corresponding to the fiber genes, and a 876 bp-long Ad5fragment corresponding to the Ad5 E4 region (located immediatelydownstream of Ad5 fiber gene) were purified by agarose gelelectrophoresis. To generate chimeric DNA probes, amplified Ad5 DNA wasmixed with Ad9 or Ad35 fragments obtained during the first step of PCR,and subjected to a second PCR amplification using Ad5/9F or Ad5/35Fprimers and the Ad5R1 primer. The resulting Ad5/9 or Ad5/35 chimeric DNAfragments (see FIG. 15) were purified and their concentrations weremeasured spectrophotometrically. Corresponding chimeric DNA fragmentswere loaded as concentration standards on agarose gels or labeled with[³²P]-dCTP and used as probes for Southern analysis. The number of viralgenomes per DNA sample was calculated after quantitative Phospho-imageranalysis. In preliminary experiments, no preferential hybridization ofchimeric DNA probes to DNA of any particular viral serotype wasdetected.

Results

Replication of Selected Serotypes in K562 and CD34+ Cells:

Adsorption/internalization studies do not ultimately prove viraltransduction, a process often defined as gene transfer that allows forviral or heterologous gene expression in host cells. Intracellulartrafficking, including endosomal lysis, transport to the nucleus, andnuclear import of the viral genome, depends on structural capsidproteins and thus, varies between different serotypes (Defer, C., etal., P. 1990. J. Virology. 64:3661–3673; Miyazawa, et al. 1999. J.Virology. 73:6056–6065). We believed that analysis of viral geneexpression would be a means to verify successful nuclear import of viralgenomes and that this would be a good criterion for selection ofserotypes able to efficiently infect our target cells. To do this, weused a protocol, which allows for the detection of Ad replication ininfected cells. Viral DNA synthesis can only occur after de novoexpression of adenoviral early genes. We utilized a site-specificmethylation strategy to monitor viral DNA replication within infectedcells, (Nelson, J., Kay, M. A. 1997. Journal of Virology. 71:8902–8907).Methylated Ad serotypes were produced by the addition of a methyl grouponto the N6 position of the adenine base of Xho I sites, CTCGAG, duringpropagation of the viruses in 293 cells expressing the Xho Iisoschizomer PaeR 7 methyltransferase (PMT) (Kwoh, T. J., et al. 1986.Proc. Natl. Acad. Sci. USA. 83:7713–7717) (293 PMTcells). Loss ofmethylation through viral replication restores Xho I cleavage and can bedetected by Southern blots of Xho I-digested genomic DNA from infectedcells.

Ad replication studies were performed in K562 and CD34+ cells with Ad9and Ad35, in comparison to Ad5. For replication studies, the infectioustiter (in pfu/ml) and genome titer (in genomes per ml) were determined(by plaque assay on 293 cells or by quantitative Southern blot,respectively) for methylated and unmethylated Ad5, Ad9, and Ad35 (Table2). The ratio of pfu to genome titer was comparable for methylated andunmethylated virus demonstrating that DNA methylation had not alteredtransduction properties. About 85% of (Ad5, 9, and 35) virus used forinfection was methylated as calculated based on the intensity offragments specific for methylated and non-methylated viral DNA presentin the viral load (FIG. 15). The numbers of genomes detected afteradsorption (1 hour, 0° C.) or internalization (2 hours 37° C.)correlated well with studies shown in FIG. 12. Ad9 and Ad35 interactedmore efficiently than Ad5 with K562 and CD34+ cells. Dose-dependentreplication studies in K562 and CD34+ cells were performed with the samegenome numbers of Ad5, 9, and 35 (FIG. 15). The replication rate wasmeasured based on the ratio of methylated to demethylated viral DNAafter infection with different MOIs (2100, 420, and 105 genomes percell). In K562 cells, efficient replication (100% conversion frommethylated to unmethylated DNA) was detected for Ad5 at MOI>/=2100, forAd9 at MOI>/=420, and for Ad35 at MOI>/=105. This demonstrated that Ad35transduced K562 cells with the highest efficiency. In CD34+ cells, thereplication rate was 100% for Ad5 and 31% for Ad9 after infection withMOI 420. Although methylated Ad35 viral DNA was present in CD34+ cells,viral replication was undetectable for Ad35. In summary, while viralreplication studies in K562 cells confirmed data obtained for Ad5, 9,and 35 adsorption and internalization, there was a discrepancy betweenearlier results and the poor replication of Ad9 and, particularly, Ad35in CD34+ cells. As outlined later, replication analysis in heterogeneouscell populations, like CD34+ cells, may not allow for definitiveconclusions on tropism of a particular serotype.

Taking all the screening data together, Ad9 and Ad35 emerged as thevariants with the strongest tropism for K562 and CD34+ cells. It isthought that Ad9 can bind to CAR, however, it preferentially usesα_(v)-integrins for cell entry (Roelvink, P. W., et al. 1996. JVirology. 70:7614–7621). This entry strategy may be not optimal forefficient infection of CD34+ cells as only less that 17% of them expressα_(v)-integrins (FIG. 10). Therefore, we decided to concentrate on Ad35as a source for heterologous fiber to be used for construction of achimeric vector based on an Ad5 backbone.

TABLE IV Results from the infectivity assay which determines the opticalparticle-to-PFU (OPU/PFU) ratio using 293 cells Virus OPU (A260) PFUOPU/PFU ratio Ad5 1.4 × 10¹² 1.06 × 10¹¹ 13 Ad9 4.61 × 10¹¹ 2.6 × 10⁸1773Discussion

Viral replication studies in K562 cells confirmed the data obtained forAd5, 9, and 35 adsorption and internalization. However, there was adiscrepancy between the interaction data and the replication data inCD34+ cells where Ad9 replicated only poorly and no replication was seenfor Ad35. Ad replication is only initiated upon the production of acritical threshold of early viral proteins, which in turn, is directlydependent on the number of viral genomes present in the nuclei ofinfected cells. Therefore, the outcome of replication studies may beaffected by the rate of nuclear import of viral genomes, by the activityof viral promoters, and/or the intracellular stability of viral DNA/RNA.These parameters may vary, on one hand, between different subsets ofCD34+, and/or, on the other hand, between different Ad serotypes. Inconclusion, the viral replication analyses performed with different Adserotypes in CD34+ cells may not predict the actual transductionproperties of chimeric vectors based on Ad5 backbone. This implies thatattempts to produce gene transfer vectors based on Ad genomes other thanAd5 should be exercised with caution.

Recently, an Ad serotype screening strategy was used to identifyvariants with tropism for primary fetal rat CNS cortex cells or humanumbilical vein endothelial cells. The optimal serotype (Ad17) wasselected based on immunohistochemistry for hexon production 48 hoursafter infection (Chillon, M., et al. 1999. J. Virology. 73:2537–2540).However, this approach is problematic because, at least in our hands,antibodies developed against Ad5 hexon did not cross-react with otherserotypes. Also, hexon is expressed only after onset of replication. Asoutlined above, the kinetics of intracellular trafficking, viral geneexpression, and replication significantly vary between serotypes (Defer,C., et al., P. 1990. J. Virology. 64:3661–3673; Miyazawa, et al. 1999.J. Virology. 73:6056–6065).

In addition to being the most efficient serotype in terms of interactionwith CD34+ cells, Ad35 is also interesting because it interacts withreceptor/s different from the Ad5 and Ad3. Ad35 and Ad5GFP/F35attachment was not inhibited by Ad5 or anti-CAR antibodies suggestingthat Ad35 binding was CAR independent. First, Ad5 did not compete withAd35 and Ad5GFP/F35 during internalization and infection indicating thatα_(ω)β_(3/5□) integrins are not involved in viral entry. Second,function-blocking antibodies against α_(v)-integrins did not competewith Ad35 and Ad5GFP/F35 for internalization into K562 cells, whereasthese antibodies did inhibit Ad5 internalization. And third, in contrastto Ad5 based vectors, GFP expression after infection with Ad5GFP/F35 wasnot restricted to α_(v)-integrin-expressing CD34+ cells. From thesefacts, we conclude that infection with Ad35 and the chimeric Ad5GFP/F35vector does not involve α_(v)-integrins. In this context, the presenceor absence of RGD motifs within Ad35 penton base remains to bedetermined by sequencing the corresponding genome region.Cross-competition assays demonstrated that Ad35 and Ad5GFP/F35 bind to areceptor that is different from the Ad3 receptor. Although Ad3 and 35belong to the same subgroup, they have been divided into two DNAhomology clusters, B1 and B2; the amino acids composing their fibers areonly 60% homologous. Furthermore, the target tissues for both virusesare different; Ad3 can cause acute respiratory infections, whereas Ad35is associated with kidney infection (Horwitz, M. S. 1996. Adenoviruses,p. 2149–2171. In B. N. Fields, Knipe, D. M., Howley, P. M. (ed.),Virology, Vol. 2. Lippincott-Raven Publishers Inc., Philadelphia).Therefore, it was not surprising to see that Ad3 and Ad35 recognizedifferent receptors.

In conclusion, Ad35 and the chimeric vector enter the cells by a CAR-and α_(v)-integrin independent pathway. We believe that Ad35 and thechimeric vector binds primarily to its fiber receptor and that thisinteraction is sufficient to trigger internalization. On the other hand,Ad35 internalization may involve cellular proteins other thanα_(v)-integrins. These membrane proteins can overlap with those for Ad3internalization and represent β2 integrins, which protrude more from thecell surface than α_(v)-integrins (Huang, S., et al. 1996. J. Virology.70:4502–4508).

According to EM studies of negative contrast-stained adenoviralsuspensions, the percentage of deficient particles for all adenoviralserotypes tested does not exceed 5%. However, plaque assays reveal thatthe ability to form plaques in 293 cells is significantly different fortested serotypes. The optical particle-to-PFU (OPU/PFU) ratio obtainedis 13 for Ad5, which is in good agreement with the previously estimatedratio for this adenoviral serotype. Importantly, this ratio is aboutthree times higher for adenovirus serotype 35 and more than 150-foldhigher for adenovirus serotype 9. Furthermore, quantitative Southernblot using chimeric Ad5/9 and Ad5/35 DNA probes is used to determine theratio between the genome and transducing titer. This study confirms thedata obtained by plaque assay. Quantitative replication assay of theseadenoviruses in K562 and CD34+ cells also confirms the ability of Ad9and Ad34 to more efficiently attach to these cell types. The replicationof viral genomes is observed for Ad9 and Ad34 at lower MOIs ofinfection, compared to Ad5. In conclusion, the data obtained fordifferent serotypes in attachment and internalization are in goodagreement with the infectivity data in target cells.

E. Attachment and Internalization of Different Adenoviral Serotypes intoPrimary Dendritic Cells, JAWSII, MCF-7 and REVC Cells.

As a proof of principle, the serotype screening strategy can be employedfor other important target cells which are refractory to Ad5 infection.

Results

RECV cells are endothelial cells which have to be targeted forapproaches that are aimed to gene therapy of restenosis,atherosclerosis, inflammation etc. MCF-7 cells are breast cancer cellsisolated from liver metastases which are important targets for tumorgene therapy. The human adenovirus serotypes 3, 5, 9, 35 and 41 aretested to see whether they can attach to and can be internalized bymouse primary dendritic cells, JAWSII cells, MCF-7-human breast cancercells and REVC endothelial cells. None of the adenoviral serotype testedcan efficiently attach to primary dendritic cells. Adenovirus serotype 3is able to efficiently attach to REVC endothelial cells (about 400 virusparticles per cell are attached and about 300 are internalized). Incomparison, only 50 Ad5 particles are able to attach to and even fewerare internalized in these REVC. The human breast cancer cells (MCF-7)are previously shown to be refractory to Ad5 infection at low MOIs.However, Ad3 and more efficiently, Ad35 attach to and internalize intoMCF-7 cells.

Discussion

The data presented herein indicate that different human adenovirusserotypes recognize different cellular receptors and can thereforeinfect cell types that are refractory to Ad5 infection. There areadenoviral serotypes that can more efficiently attach and internalizethan Ad5 for human CD34+ cells, REVC, K562 and MCF-7 cells. This findingprovides a basis for the construction of chimeric adenoviral vectorswhich are Ad5 vectors containing receptor ligands derived from otherserotypes.

F. Infection Studies on Primary Human Bone Marrow Cells.

Since established erythroleukemic cell lines do not represent anadequate model for the ultimate hematopoietic stem cell that has to betargeted in patients in order to achieve long-term reconstitution withgenetically modified cells, normal primary human bone marrow cells areused for the initial infection/retargeting studies.

Results

In a first set of tropism studies with different Ad serotypes, wholebone marrow cell suspensions can be used without preselection. This isadvantageous because the tropsim of various adenovirus serotypes orgenetically retargeted vectors can be analyzed on a broad spectrum ofprogenitor subpopulations representing myeloid, erythroid,megakaryocytic, lymphoid, dentritic, and monocytic lineages. For shortterm (<5 hours) infection studies, bone marrow suspensions can becultured in IMDM supplemented with 10% FCS, β-mercaptoethanol, and 10u/ml IL-3 for ensuring cell viability.

Mononucleated Cell Assays:

Mononucleated bone marrow cells can be incubated with MOI 1, 10, 100, or1000 pfu/cell of the various adenovirus types for a short time. Paraffinsections or cytospins of infected bone marrow cells can be analyzed fornuclear-localized, labeled viral DNA. BrdU labeling can be visualized byimmunoflouresence with anti-BrdU antibodies; ³²P-tagged viral DNA can bedetected by incubation with photo-emulsion. In addition, the same cellmaterial can be analyzed for morphology after specific histo-staining(e.g. Wright, Hemo3 staining). If required, commercially availableantibodies can be used to specific cell surface markers conjugateddirectly to different fluorochromes (FITC (green), TRIT., RPE, (red),RPE-Cy5, AMCA (blue)) to completely characterize infected bone marrowsubpopulations. Colocalization of BrdU-labeled viral DNA (e.g. as FITCsignal) with membrane markers signifying infection of specific celltypes can be demonstrated; for example, potential stem cells/earlyprogenitors (CD34⁺, CD38⁻), megakaryocytes (CD41a+), eryhthroid cells(glycophorin A+), dentritic cells (CD1a+), monocytes (CD14+), or myeloidcells (CD15+), etc. The morphological analysis of infected bone marrowsubsets gives a first information whether specific adenovirus serotypescan target primitive cell types.

Discussion

Since the different wild-type adenoviruses do not express a uniformmarker gene and do not integrate and since detection of tagged viral DNAcannot be done on live cells, it is not possible, at this point, tocharacterize infected cells for clonogenic or repopulation capacities.Therefore, adenovirus serotypes for retargeting studies are selected,based on their ability to infect in vitro purified CD34+ cells at lowMOIs. This subset of bone marrow cells is known to contain long-termreconstituting cells. Infection studies with different adenovirusserotypes can be repeated on purified CD34+ cells, (cultured in IMDM+10%FCS, α-mercaptoethanol, and 10 units/ml IL-3) as described above.Purification of CD34+ cells can be performed by direct immunoadherenceon anti-CD34 monoclonal antibody-coated plates or on MiniMacs columns asdescribed by Papyannopoulou (Papayannopoulou, T. et al., 1996,Experimental Hematology, 24:660–69; Papayannopoulou, T. et al., 1993,Blood, 81:229). The purity of isolated CDC34+ cells ranges routinelyfrom 80–95%. Analgous infection studies can be repeated with selectedadenovirus types on CD34+/CD38− subsets.

To confirm productive infection purified CD34+ cells can be infectedwith selected (methylase-tagged) serotypes and analyze viral DNAreplication. Cultures of purified human bone marrow CD34+ cells can beused for the transduction and integration studies as a model for HSCs.

It was recently demonstrated that HSC activity does exist inCD34-negative human bone marrow subsets (Bathia, M. et al., 1998, NatureMedicine, 4; 1038–45; Osawa, M., et al., 1996, Science, 273:242–5;Goodell, M. et al., 1997, Nature Medicine, 3:1337–45; Zanjani, E. D. etal., 1998, Exp. Hematology, 26:353–60). Lin ⁻CD34⁻38⁻ cells can betested in the retargeting and transduction studies in combination withrepopulation assays in SCID-NOD mice.

G. Cloning and Insertion of the Fiber Gene.

Methods

PCR-Cloning of the Corresponding Fiber Gene and Insertion into Ad5 BasedShuttle Plasmids Instead of the Endogenous AD5 Fiber:

One or several adenoviruses with tropism to CD34+ or other HSCcontaining population is selected for further studies described herein.The complete coding region for fiber varies between 1–2 kb, depending onthe virus type. The fiber encoding sequences can be obtained by PCR withPfu polymerase from viral DNA isolated from purified particles of theselected virus types. The corresponding primers can be designed based onthe fiber sequences available from the EMBL gene bank. The PCR productsare cloned as PacI-BalI fragment into pCD4 (FIG. 10), a shuttle vectorfor recombination of RecA+E. coli. In pCD4, the heterologous fiber geneis flanked on both sides with Ad5 sequences, which are homologous toregions directly adjacent to the fiber reading frame in Ad5. As an Ad5(shuttle vector) derived template for recombination, pCD1, a pBHG 10(Microbix, Toronto, Canada) derivative can be used. The recombinationprocedure is performed according to a protocol routinely used forrecombinant adenovirus generation (Chartier, C., et al., 1996, J. ofVirology, 70, 4805–4810). Routinely, 90% of the resulting plasmids areaccurately recombined. The junctions between the heterologous fiber (X)and Ad5 sequences can be sequenced to confirm the accuracy ofrecombination. The resulting plasmid is named pAd5fiberX (pAd5^(fx)).The resulting product is used to generate pAd5^(fx)-based Ad.AAVcontaining the heterologous fiber gene.

Construction of Chimeric Ad Vectors:

For transduction studies, two Ad vectors were constructed: Ad5GFP andAd5GFP/F35, containing a chimeric Ad5/35 fiber gene. Both adenoviralvectors contained a 2.3 kb, CMV promoter driven EGFP gene [derived frompEGFP-1, (Clontech, Palo Alto, Calif.)] inserted into the E3 region ofAds. The EGFP expression cassette was cloned between Ad5 sequences25,191–28,191 and 30,818–32,507 into a shuttle plasmid, which containedthe E3 deletion described for pBHG10 (Microbix, Toronto, Canada). Theresulting plasmid was named pAdGFP. For the chimeric vector, the Ad5fiber gene in pAdGFP was substituted by an Ad5/35 chimeric fiber genegenerated by the two-step PCR protocol outlined above. In the first PCRstep, three DNA fragments corresponding to i) the Ad5 fiber5′-nontranslated region and the first 132 bp of the fiber tail domain(nt 30,818–31,174), ii) the Ad35 shaft and knob domains (nt 132–991),and iii) the Ad5 E4 region including the Ad5 fiber polyadenylationsignal (nt 32,775–33,651 were amplified by Pfu-Turbo DNA polymerase. Thefollowing primers were used: for the Ad5 tail, Ad5F-2 (nt 30,798–30,825)5′-CGC GAT ATC GAT TGG ATC CAT TAA CTA-3′ (SEQ ID NO.: 9) and Ad5R-2 (nt31,174–31,153) 5′-CAG GGG GAC TCT CTT GAA ACC CAT T-3′ (SEQ ID NO.: 10);for the Ad35 shaft and knob, primers Ad5/35F and Ad5/35R (see above);for the Ad5E4 and polyA, primers Ad5F-1 and Ad5R-1 (see above). After 10PCR cycles, the products were purified by agarose gel electrophoresis,combined, and then subjected to a second PCR with primers Ad5F-2 andAd5R-1. The resulting 2115 bp-long chimeric fiber gene contained the Ad5tail and the Ad35 shaft and knob domains. This product was used as asubstitute for the SalI/XbaI Ad5 fiber gene containing fragment inpAdGFP. The resulting plasmid was named pAdGFP/F35. To generatefull-length E1/E3 vector genomes, pAdGFP and pAdGFP/F35 were inserted inpAdHM4 (Mizuguchi, H., Kay, M. A. 1998. Human Gene Therapy. 9:2577–2583)by recombination in E. coli (Chartier, C., E. et al. 1996. Journal ofVirology. 70:4805–4810). To do this, the RecA+E. coli strain BJ5183 wasco-transformed with pAdHM4 linearized by SrfI mixed with the XbaIfragments containing the GFP genes, the Ad5 or Ad5/35 fiber genes, andthe Ad5 homology regions. The resulting recombinants were analyzed byrestriction analysis. Correct recombinants were amplified in E. coliHB101 and purified by double CsCl gradient banding. The plasmids werenamed pAd5GFP and pAd5GFP/F35. The correct structure of the Ad5/35chimeric fiber gene was confirmed by endonuclease digestion andsequencing part of pAd5GFP/F35. To produce the corresponding viruses,pAd5GFP and pAd5GFP/F35 were digested with PacI to release the viralgenomes and transfected onto 293 cells as described (Lieber, A., C.-Y.et al. 1996. Journal of Virology. 70:8944–8960). Plaques developed 7 to10 days post-transfection in overlayed cultures. Recombinant viruseswere propagated in 293 cells and purified by standard methods describedelsewhere (Lieber, A., C.-Y. et al. 1996. Journal of Virology.70:8944–8960).

Hemagglutination Assay:

Twenty-five microliters of serial dilutions of Ad5, Ad35, or chimericAd5GFP/F35 virions in McIlvaine-NaCl buffer (0.1 M citric acid, 0.2 MNa₂HPO₄ [pH 7.2], diluted 1:50 with 0.87% NaCl) were loaded onto 96 wellplates. To each dilution, 25 μl of a 1% suspension of monkeyerythrocytes (in McIlvaine-NaCl buffer) was added. The sedimentationpattern was determined after incubation for 1 hour at 37° C. All testswere performed in quadruplicates in at least two independentexperiments.

Southern Blot:

Extraction of genomic DNA, labeling of DNA fragments and hybridizationwere performed as described earlier (Lieber, A., C.-Y. et al. 1996.Journal of Virology. 70:8944–8960).

Results

Construction/Characterization of Chimeric Fiber:

Previously, it has been shown that exchanging the fiber knob wassufficient to alter the tropism of chimeric Ad vectors (Chillon, M., etal. 1999. J. Virology. 73:2537–2540; Krasnykh, V., et al. 1998. J.Virology. 72:1844–1852; Stevenson, S. C., et al. 1997. J. Virology.71:4782–4790). As outlined above, the length of the fiber shaft maycritically determine the entry strategy of a particular serotype.Therefore, we decided to replace not only the Ad5 fiber knob but alsothe shaft. The chimeric Ad5/35 fiber contained the Ad5 tail (amino acid:1–44) necessary for interaction with the Ad5 penton base linked to 279amino acids from Ad35 including the shaft with 7β-sheets and the knob(FIG. 16A). The endogenous Ad5 fiber polyA signal was used to terminatetranscription of the chimeric fiber RNA. The combination of the Ad5capsid including the RGD motif containing penton base with ashort-shafted fiber could be risky because the natural distance betweenthe fiber knob and the RGD motifs was disturbed. The Ad5 fiber wassubstituted by the chimeric fiber sequences based on an E1/E3 deleted Advector. This vector carried a CMV promoter-GFP reporter gene cassetteinserted into the E3 region. The corresponding chimeric virus(Ad5GFP/F35) was produced in 293 cells at a titer of >2×10¹² genomes perml. For comparison, an E1/E3 deleted Ad vector containing the originalAd5 fiber gene and the GFP expression cassette was generated (Ad5GFP).The titer and the ratio of physical to infectious particles was similarbetween Ad5GFP and Ad5GFP/F35 indicating that the fiber modification didnot significantly alter the stability and/or growth properties of thechimeric vector. The correctness of the fiber modification was confirmedby restriction analysis of the Ad5GFP/F35 viral genome followed bySouthern blot hybridization (FIG. 16B), direct sequencing of thefiber-coding region, and a functional test for hemagglutination (HA) ofmonkey erythrocytes. The agglutination of erythrocytes is fiberknob-mediated; it is known that Ad5 does not agglutinate monkeyerythrocytes whereas Ad35 efficiently does (Pring-Akerblom, P., et al.1998. J. Virology. 72:2297–2304). In HA tests, Ad5GFP/F35 agglutinatedmonkey erythrocytes with the same efficiency as Ad35 at dilutions of upto 1:512. In contrast, no hemagglutination was observed with equivalentAd5 dilutions. This clearly confirmed the functional activity of thechimeric Ad5/35 fiber incorporated into Ad5 capsid.

Generation of chimeric adenoviral vectors (AD.AAV^(fx)) withheterologous fiber molecules: Adenoviruses with chimeric Ad5-Ad3 fiberare viable and can be produced at high titers (Krasnykh, V., et al.,1996, J. of Virology, 70, 6839–6846; Stevenson, S. C. et al., 1997, J.Virology, 71:4782–90). In order to test whether the fiber substitutiondescribed herein affects production or stability of adenoviruses, twoE1-deleted first-generation, adenoviral vectors are produced with theAAV-βgal cassette in 293 cells using standard protocols. The vector isgenerated by recombination of pAd.AAV-BG (prepared as in FIG. 17) withpCD1 (containing the endogenous Ad5 fiber) (FIG. 9); the other vector(with heterologous fiber) is the recombination product of pAd.AAV βgaland pAd5fiberX (pAd5^(fx)) (FIG. 9). Virus from single plaques isamplified on 293 cells. The production yield per 293 cell can bedetermined by plaque-titering of 293 cell lysates. It is anticipatedthat the fiber modification will not critically effect the stability ofchimeric vectors. Finally, bone marrow cells can be infected with theretargeted vectors. Two days after infection, live-cell cytometry isperformed for β-gal expression using as substrate Fluouresceindi-β-D-Galactopyranoside (FDG) (Cantwell, M. J. et al., 1996 Blood 88,4676–4683; Neering, S. et al., 1996, Blood, 88:1147–55; Fiering, S. N.et al., 1991, Cytometry, 12:291; Mohler, W. et al., 1996, PNAS, 93:57)and the infected cells are characterized for morphology and surfacemarkers. Before and during infection, bone marrow cells can be culturedin IMDM/FCS supplemented with thrombopoietin (Tpo), which supports thesurvival of HSC (Matsunaga, T. et al., 1996, Blood, 92:452–61;Papayannopoulou, T. et al., 1996, Experimental Hematology, 24:660–69).Alternatively, retargeted vectors can be generated with the AAV-GFP(green fluorescence protein) cassette and perform FACS analysis ontransduced cells based on GFP and surface marker expression.

H. Competition Studies of Chimeric Fiber Protein Ad5/35.

Competition Studies:

Cross-competition studies between Ad5, 35, and Ad5GFP/F35 (FIG. 18) forbinding and internalization were performed in order to investigate inmore detail the pathways which are used by the chimeric vector to infecttarget cells. Wild-type Ad35 and the chimeric vector Ad5GFP/F35 couldrecognize the same primary receptor as they competed with each other forthe attachment to K562 cells (FIG. 19A, upper pane). This primaryreceptor is different from that used by Ad5, since neither Ad5 viralparticles nor anti-CAR monoclonal antibodies (FIG. 19B, upper panel)were able to abrogate Ad35 or Ad5GFP/F35 binding. In competition studiesfor internalization, Ad35 and Ad5GFP/F35 competed with each other withequal efficiency. Ad5 and anti-α_(v)-integrin monoclonal antibodies(L230) (FIGS. 19C, D; lower panel) did not inhibit internalization ofAd35 or the chimeric virus. To consolidate this data, K562 cells wereinfected with Ad5GFP and Ad5GFP/F35 after prior incubation of cells withanti-CAR or anti-α_(v)-integrins monoclonal antibodies followed byanalysis of GFP-expressing cells. The transduction data mirror theresults obtained in adsorption/internalization studies. In summary, thisdemonstrated that Ad35 and Ad5GFP/F35 use a CAR andα_(v)-integrin-independent pathway for infection of K562 cells; thestructural elements which account for these specific properties arelocated within the Ad35 fiber and can be transplanted into Ad5 by fibersubstitution.

Ad3 can efficiently interact with K562 cells (FIG. 12), although Ad3 andAd35 belong to the same subgroup (B), the homology between amino acidsequences of their fibers is only about 60%. Therefore, we decided totest whether Ad3 could compete with Ad35 and Ad5GFP/F35 for attachmentand internalization (FIG. 20). These studies demonstrated that Ad35binding was not inhibited by Ad3 indicating the use of differentreceptors. Interestingly, Ad3 slightly inhibited attachment ofAd5GFP/F35 (FIG. 20A, left panel). In addition to binding to thereceptor common for the Ad35 and Ad5GFP/F35 fiber, the chimeric capsid(e.g. the Ad5 penton RGD motifs) may also interacts with a secondcellular receptor that overlaps with elements involved in Ad3 binding.In cross-competition for internalization, pre-incubation of cells at 37°C. with Ad35 and with chimeric virus significantly decreasedinternalization of [³H]-labeled Ad3 (FIG. 20D, right panel). In thereverse experiment, Ad3 as competitor decreased the level ofinternalization by 30% for both, Ad35 and the chimeric virus (FIG. 20B,right panel). As expected, Ad5 and Ad3 did not compete for adsorption orinternalization. As shown before (FIG. 19B), anti-CAR andanti-α_(v)-integrin antibodies did not block Ad3 interaction with K652cells. In summary, we concluded that Ad35 and Ad5GFP/F35 bind toreceptor/s different from that of Ad3, although they can use commonstructural elements for internalization, which are different fromα_(v)-integrins.

Infection Studies with Chimeric Virus:

It is established that Ad5GFP/F35 infected K562 cells by a CAR andα_(v)-independent pathway. It is possible that this property allows forefficient transduction of non-cycling CD34+ cells, which expressscarcely CAR and α_(v)-integrins. To test this, the transductionproperties of Ad5GFP and Ad5GFP/F35 vectors were analyzed on CD34+cells, K562, and HeLa cells. FIG. 21 shows the percentage of transduced,GFP expressing cells depending on the MOI used for infection. Nearly100% of HeLa cells were transduced with Ad5GFP and Ad5GFP/F35 at MOIsof >/=25. More than 95% of the K562 cells were transduced withAd5GFP/F35 at MOIs of >/=100, whereas the transduction rate wassignificantly lower with Ad5 where it increased with the MOI reaching aplateau at ˜70% GFP-positive cells after infection with an MOI of 400.Transduction of CD34+ cells was about three fold more efficient withAd5GFP/F35 than with Ad5GFP at all MOIs analyzed. Interestingly, athigher MOIs, the transduction rate did not rise proportionally with theviral dose and soon reached a plateau indicating that in both cases onlyspecific subset/s of CD34+ cells were permissive to infection. In orderto characterize in more detail these specific, permissive subset/s,additional transduction studies were performed. First, the percentage ofGFP expressing cells was determined in CD34+fractions that were stainedfor α_(v)-integrins or CARs (FIG. 22). The low number of CAR positiveCD34+ cells complicated accurate co-labeling studies, and there was nocorrelation between CAR expression and the proportion of transducedcells among CD34+ cells infected with Ad5GFP or Ad5GFP/F35.Interestingly, for Ad5GFP, 65% of all GFP expressing cells were positivefor α_(v)-integrins, whereas less than 22% of GFP positive cellsinfected with the chimeric virus stained positive for α_(v)-integrinexpression. While only 17% of the whole CD34+population expressed GFPafter Ad5GFP infection, the percentage of GFP-expressing cells in theCD34+/□_(v)-integrins positive fraction was 50%. This indicates thatAd5GFP vector-mediated GFP expression was preferentially localized toα_(v)-integrin positive CD34+subsets, whereas after infection with theAd5GFP/F35 vector, GFP was expressed in a broader spectrum of CD34+cells with most of them being α_(v)-integrin-negative.

Next, transduced cells were simultaneously analyzed for GFP as well asfor CD34 and CD117 markers. As mentioned before, only about 90% of allcells used in our analysis were positive for CD34 at the time ofinfection, hence the multiparameter analysis for CD34 and GFP. Apopulation of CD34+ cells is extraordinarily heterogeneous in morphologyand stem cell capacity. The subpopulation of CD34+ and CD 117+ cellsresembles very primitive hematopoietic cells (Ikuta, K, Weissman, I. L.1992 Proc. Natl. Acad. Sci. USA. 89:1502–1506; Simmons, P. J, et al.1994. Expl. Hematology. 22.157–165). FIG. 23 summarizes the analyses ofGFP expression in correlation with these specific stem cell markers.While 54% of cells infected with chimeric vector were positive for GFPand CD34+, only 25% of cells infected with Ad5GFP expressed thetransgene and CD34+marker (FIG. 23A, lower panel). More importantly,based on GFP expression, the chimeric virus transduced 80% of c-kitpositive cells, whereas the Ad5-based vector transduced only 36% (FIG.23A, middle panel). In an additional experiment, CD34+ cells were sortedfor CD117 expression prior to infection with Ad5GFP or Ad5GFP/F35 and,24 hours post-infection, GFP expression was analyzed in this specificfraction (FIG. 23B). This analysis revealed that the chimeric vectorstransduced 4 fold more CD34+/CD117+ than the Ad5GFP vector.

In conclusion, these results demonstrated that the chimeric Ad5GFP/F35vector was clearly superior to the Ad5GFP vector in targeting andtransduction of CD34+ cells. Furthermore, the data suggest that thespectrum of CD34+ cell subsets permissive for Ad infection wassignificantly different for the chimeric vector than for the Ad5 vector.

Analysis of Viral Genomes within CD34+ Cells Infected with the Ad5 andChimeric Vectors:

So far, the transduction rate of CD34+ cells was measured based on GFPexpression after infection with Ad5GFP and Ad5GFP/F35. Considering theextraordinary heterogeneity of CD34+ cells in morphological andfunctional parameters, GFP may not be expressed in all cell types thatwere efficiently infected. Reasons for this include that the CMVpromoter may not be active in all cell types or that the regulation oftransgene expression could differ between subsets on apost-transcriptional or post-translational level. To test this, wequantified the number of intracellular (transduced) viral genomes withinGFP positive and GFP negative fractions of CD34+ cells infected withAd5GFP and Ad5GFP/F35. To do this, twenty-four hours after infection,CD34+ cells were sorted for GFP positive and GFP negative fractions,which were subsequently used to isolate genomic DNA together withtransduced viral DNA. The number of viral genomes was determined byquantitative Southern blot as described for FIG. 15. Per GFP-positiveCD34+ cell, about 270 copies of the Ad5GFP/F35 viral genome weredetected. Interestingly, a remarkable 200 copies of the Ad5GFP/F35 viralgenome were found per GFP-negative CD34+ cell (FIGS. 24A and 25). Thisdemonstrated that not all infected cells expressed GFP and implies thatthe actual transduction rate was higher than 54% (GFP-positive cells).We concluded that the CMV promoter was not active in all transducedCD34+ subsets. No Ad5GFP vector specific signal was detected withininfected CD34+ (GFP positive or negative) fractions by Southern blotwhich had a detection limit of 14 viral genomes per cell. From this, wecan conclude that the vector DNA concentration per transduced cell wasat least 20 times higher for Ad5GFP/F35 than for Ad5GFP.

Ad5GFP DNA was only detectable in DNA samples from infected CD34+ cellsby Southern blot after prior PCR amplification with vector specificprimers (FIGS. 24B and 25). This indicates that the replicationdeficient Ad5 vector is present but at a very low copy number, which maybe limited by intracellular genome stability. Using the PCR-Southerndetection method, Ad5 vector DNA was also detected in GFP negativecells, supporting that the CMV promoter may not have been the optimalchoice for transduction studies. It is notable that studies by others onviral genome analyses after infection of CD34+ cells with Ad5 vectorswere performed only after prior PCR amplification (Mitani, K., et al.1994. Human Gene Therapy. 5:941–948; Neering, S. J., et al. 1996. Blood.88:1147–1155).

Discussion

The chimeric Ad5GFP/F35 vector has binding and internalizationproperties similar to Ad35. Therefore, the fiber substitution wassufficient to swap cell tropism from Ad5 to Ad35. The Ad5GFP/F35 capsidchimera contained the short-shafted Ad35 fiber incorporated into an Ad5capsid, instead of the naturally occurring long-shafted Ad5 fiber.During Ad5 infection, interaction between the penton base and intergrinsis required to induce viral internalization. For this interaction, thelength of fiber shaft and the precise spatial arrangement of knob andRGD motifs are critical for the virus entry strategy. The naturalspatial arrangement is disturbed when short-shafted heterologous fibersare inserted into the Ad5 capsid. Interestingly, the Ad5/35 capsidchimera allows for efficient infection, suggesting that the protrudingRGD motives in the Ad5 penton base do not affect the interaction withthe primary Ad35 receptor. So far, most of the chimeric viruses weregenerated by substituting only the Ad5 knob while maintaining the longAd5 fiber shaft (Chillon, M., et al. 1999. J. Virology. 73:2537–2540;Krasnykh, V. N., et al. 1996. J. Virology. 70:6839–6846; Stevenson, S.C., et al. 1995. J. Virology. 69:2850–2857; Stevenson, S. C., et al.1997. J. Virology. 71:4782–4790). The exception was an Ad5/7 chimericvirus (Gall, J., et al. 1996. J. Virology. 70:2116–2123), where thewhole Ad5 fiber was substituted by the short-shafted Ad7 fiber. However,similar to the parental Ad5, the Ad5/7 chimera still requiredα_(v)-integrins for infection.

This Ad5GFP/F35 chimera is the first demonstration that despite thepresence of RGD motifs within the Ad5 penton, the chimeric virus usescell entry pathways determined primarily by the receptor specificity ofthe short-shafted heterologous fiber. This does not exclude thatinteraction with a secondary receptor may increase binding affinity. Thelatter is supported by the observation that Ad35 and Ad5GFP/F35 slightlydiffered in their ability to compete with Ad5 or Ad3 for binding. It ispossible that Ad5/35 attachment involves, in addition to the highaffinity fiber binding, interaction between Ad5 capsid proteins (e.g.RGD motifs) and secondary receptor/s that overlap with those used by Ad3and Ad5.

This data indicate that infection with Ad5-based vectors is restrictedto a specific subset of CD34+ cells. The percentage of GFP expressingcells after Ad5GFP infection of CD34+ cells reached a plateau at MOIshigher than 100 indicating that only a limited fraction of CD34+ cellswas permissive to Ad5. Also, strong replication of wild type Ad5 ininfected CD34+ cells may be the result of preferential transduction of aspecific subpopulation of CD34+ resulting in a expression of early viralgenes at a level sufficient to initiate viral replication. The presenceof a specific subpopulation of CD34+ cells permissive to Ad5-vectorinfection was suggested by others (Byk, T., et al. 1998. Human GeneTherapy. 9:2493–2502; Neering, S. J., et al. 1996. Blood. 88:1147–1155).In the present report, we further characterized this subpopulation anddemonstrated that Ad5-based vectors preferentially infectedα_(v)-integrin positive CD34+ cells. Integrins (including α_(v)) arethought to be important for homing and trafficking of transplantedhematopoietic cells, however little is known about the correlationbetween α_(v)-integrin expression and the differentiation status ofhematopoietic cells (Papayannopoulou, T., Craddock, C. 1997. ActaHaematol. 97:97–104; Roy, V., Verfaillie, C. M. 1999. Exp. Hematol.27:302–312). There was no clear correlation between CAR and GFPexpression suggesting that Ad5GFP may be able to use another membraneprotein as a primary receptor. Alternatively, Ad5GFP transductionobserved at an MOI of 200–400 could be the result of direct interactionbetween virus and α_(v)-integrins triggering internalization, which maybe the preferred pathway in the absence of CAR (Legrand, V., et al.1999. J. Virology. 73:907–919). Importantly, infection with the chimericAd5GFP/F35 vector was not restricted to the α_(v)-positive CD34+subpopulation.

Among CD34+ cells, the subpopulation of CD34+ and CD117+ cells resemblesvery primitive hematopoietic cells (Ikuta, K., Weissman, I. L. 1992Proc. Natl. Acad. Sci. USA. 89:1502–1506; Simmons, P. J., et al. 1994.Expl. Hematology. 22:157–165). The receptor for stem cell factor, CD117(c-kit) belongs to a tyrosine kinase family. It was previously shownthat c-kit+, CD34+ cord blood cells contain a high fraction (16%) ofhematopoietic progenitors (Neu, S., et al. 1996. Leukemia Research.20:960–971). Early in ontogeny 34+/CD117+ cells have long-termrepopulating activity (Sanchez, M. J., et al. 1996. Immunity.5:513–525). An average of 50–60% of CD34+ cells are reported to be CD117positive (Ikuta, K., Weissman, I. L. 1992 Proc. Natl. Acad. Sci. USA.89:1502–1506; Neu, S., et al. 1996. Leukemia Research. 20:960–971;Simmons, P. J., et al. 1994. Expl. Hematology. 22:157–165). In ourstudies, the chimeric vector expressed GFP in 54% CD34+ cells and 80% ofCD34+/c-kit+ cells. The actual viral transduction rate could be evenhigher because transduced Ad5GFP/F35 vector DNA was also found inGFP-negative fractions of infected cells. This indicates that the CMVpromoter used to drive GFP expression in our vectors was not active inall transduced cells. We selected the CMV promoter for transgeneexpression based on published data demonstrating that PGK and CMVpromoters allowed for efficient transgene expression in CD34 cellswhereas the HTLV-I and RSV promoter were almost inactive (Byk, T., etal. 1998. Human Gene Therapy. 9:2493–2502; Case, S. S., et al. 1999.Proc. Natl. Acad. Sci. USA. 96:2988–2993). On the other hand, studies byWatanabe et al. (Watanabe, T., et al. 1996. Blood. 87:5032–5039) suggestthat the CMV promoter is not active or rapidly silenced in certainCD34+subsets. Our data underscore this observation. Consideringretroviral transduction studies, the retroviral MLV promoter may havebeen a better candidate for transduction studies in hematopoietic cells(Bregni, M., et al. 1998. Gene Therapy. 5:465–472).

After having demonstrated that the Ad5GFP/F35 vector efficientlytransduced cells carrying stem cell specific markers, the next logicalstep would be to perform colony assays with pre-sorted GFPpositive/negative cells. However, this assay is complicated by the factthat infection with first generation Ad vectors is cytotoxic and affectsthe formation and growth of progenitor colonies in MC-cultures (Mitani,K., et al. 1994. Human Gene Therapy. 5:941–948; Watanabe, T., et al.1996. Blood. 87:5032–5039). This side effect is caused by the expressionof Ad proteins within transduced cells (Lieber, A., C.-Y. et al. 1996.Journal of Virology. 70:8944–8960; Schiedner, G., et al. 1998. NatureGenetics. 18:180–183; Yang, Y., et al. 1994. Proc. Natl. Acad. Sci. USA.91:4407–4411). Some of these proteins (e.g E4-orf4, pTP, or E3-11.6k)have pro-apoptotic activity (Langer, S. J., Schaak, J. 1996. Virology.221:172–179; Lieber, A., et al. 1998. J. Virology. 72:9267–9277;Shtrichman, R., Kleinberger, T. 1998. J. Virology. 72:2975–2983;Tollefson, A. E., A et al. 1996 J. Virology. 70:2296–2306). Clearly,this would affect the outcome of transduction studies with Ad5GFP/F35,which allows for the efficient transfer of viral genomes into CD34+cells implying significant expression of viral proteins. Moreover,recently published data indicate that short-term colony assay mostlymeasure mature progenitors and do not represent a rigorous test fortransduction of potential stem cells.

A definitive demonstration that Ad5GFP/F35 based vectors can transduceHSC requires colony assays or preferably, repopulation assays inSCID-NOD mice. We can perform these studies with gutless vectors(Steinwaerder, D. S., et al. 1999. J. Virol 73:9303–13) and integrating□Ad.AAV vectors devoid of all viral genes (Lieber, A., et al. 1999. JVirol 73:9314–24) generated based on Ad5GFP/F35 chimeric capsids.Alternatively, gutless, retargeted vectors could be used to transientlyexpress a retroviral receptor on CD34+ cells to increase theirsusceptibility to infection with retroviral vectors based on an approachthat we have published earlier (Lieber, A., et al., 1995. Human GeneTherapy. 6:5–11).

Our finding that Ad5GFP/F35 can efficiently transduce hematopoieticcells with potential stem cell capacity represents an important steptowards stable gene transfer into HSCs and gene therapy of blooddisorders. Furthermore, the virological aspects of this inventioncontribute to a better understanding of adenovirus cell interactions.

I: Retargeting of Ad5 Based Vectors with Modified Fibers CarryingSpecific Ligand Peptides for HSC and Other Cell Types

Another alternative to make Ad5-capsid-based vectors suitable for HSCgene therapy is to incorporate the coding sequence for HSC specificpeptides into the H1 loop region of the Ad5 fiber gene. The modificationof the H1-loop was successfully exercised by Krasnykh et al. with a 7amino-acid long FLAG peptide (DYDDDDK) (SEQ ID NO.: 11). Using phagedisplay peptide libraries (Pascqualini, R. et al., 1996, Nature,380:364–66), Renata Pasqualini (La Jolla Cancer Research Center)reported recently, at the First Meeting of the American Society for GeneTherapy, the identification of small peptide ligands specific for bonemarrow cells. The corresponding sequences encoding these peptides can beadded to modify the H1 loop sequence employing site-directedmutagenesis. Optimally, the ligands should allow for the efficientinternalization of adenoviral particles based on a CAR- and integrinindependent pathway. Modified adenoviral vectors containing the AAVBGcassette can be produced and tested for HSC tropism as described above.

Adenovirus Peptide Display:

If order to retarget adenoviruses to any cell type of interest, astrategy is provided which involves creating a library of adenovirusesdisplaying random peptides in their fiber knobs as ligands and screeningthis library for adenovirus variants with tropism to a particular celltype in vitro and potentially in vivo.

The development of the adenovirus peptide display technique is based onthe following ideas. (i) Although the tertiary structure of the Ad5fiber knob is known, it remains unclear which domains are involved inreceptor binding. There are data suggesting that receptor-bindingdomains partially overlap with hemagglutination domains, which are wellcharacterized for a number of serotypes. Therefore, three intramolecularloop regions representing potential receptor binding sites can besubstituted by random peptide libraries. Eight amino acid residues inthe center of the FG, or GH loops can be substituted by octameric randompeptides (FIGS. 26 and 27). These substitutions will replace CAR tropismand allow for infection of refractory cell types. (ii) To synthesize theoligonucleotides encoding the peptide library a novel technique toassemble pre-synthesized trinucleotides representing the codons for all20 amino acids is employed. This avoids termination codons and assuresoptimal codon usage and translation in human cells. Synthesis of acompletely randomized library is possible with all 20 amino acids beingincorporated with the same probability and a partially randomizedlibrary with only three (in average) random amino acids substitutionsper octamer at random positions with a random amino acid to maintaincertain critical features of the tertiary knob structure whileintroducing variability. The last model is based on the distribution ofamino acids present in the hypervariable CDR 1 or 2 region ofimmunoglobulins. (iii) To maintain a representative library size ofabout 10¹⁰ different octamers per modified loop, a new cloning strategyis employed to allow for insertion of the library into the wild-type Ad5genome without introducing additional amino acids at the substitutionsite and without transformation into bacteria. This strategy is based ona “seamless” cloning technique available from Stratagen. (iv) In orderto produce the library of viruses, viral genomic DNA containing themodified fiber sequences is transfected into 293 cells without reductionof the library size. This critical step is done by conjugating the virallibrary DNA to carrier Ad5-based adenovirus via poly-lysine to assure100% transfection efficiency. This technique allows for coupling of ˜1μg of plasmid DNA (or ˜1×10¹⁰ adenoviral genomes) to 10¹⁰ viralparticles which can be used to infect 293/cre cells at an MOI of 10–100.Importantly, the carrier adenoviral genome has the packaging signalflanked by lox sites preventing the packaging of carrier viral DNA afterinfection of 293 cells that express cre recombinase (293/cre). Thishelper virus system is routinely used to produce so-called gutlessadenoviruses. Therefore, the virus progeny represents library genomespackaged into capsids containing preferentially Ad5 fibers. This isimportant for the next infection step into 293 cells at a MOI of 1 toassure a homogeneous fiber population on the capsid where the fibers areencoded by the packaged genome.

J. Production of Adenovirus Vectors with Increased Tropism toHepatocytes

An example of a G-H loop substitution to target Ad5 to hepacytes wassuccessful. Preliminary tests demonstrated that two evolutionarilyconserved regions within the malaria circumsporozoite surface protein(CS) termed RI and RII+ mediate specific interaction with hepatocytesbut not with other organs (including spleen, lung, heart and brain), norwith Kupffer cells, liver endothelial cells or with other regions of thehepatocyte membrane (Cerami, C. et al., 1992, Cell, 70:1021–33;Shakibaei, M. and U. Frevert, 1996, J. Exp. Med., 184:1699–711). Theseregions are conserved among different species including Plasmodiumberghei, P. cynomogli, and P. falciparum that infect mouse, monkey andhuman hepatocytes, respectively (Cerami, C. et al., 1992, Cell,70:1021–33; Chattejee, S. et al., 1995, Infect Immun., 63:4375–81).Peptides derived from RI (KLKQPG) (SEQ ID NO.: 12) or RII(EWSPCSVTCGNGIQVRIK) (SEQ ID NO.: 13) blocked CS binding to hepatocytesand infection by sporozoites in vivo ((Cerami, C. et al., 1992, Cell,70:1021–33; Chatterjee, S. et al., 1995, Infect Immun., 63:4375–81). R1and R11+peptides were separately inserted into Ad5-fiber knob (H-I andG-H loop) containing mutation with abolished binding to CAR and alpha-vintegrins (Kirby, L. et al., 2000, J. Virol., 74:2804–13; Wickham, T. J.et al., 1995, Gene Ther., 2:750–6). Based on preliminary data, ashort-shafted fiber was used so that the virus entry strategypredominantly depends on the interaction with the primary(hepatocyte-specific receptor). The hepatocyte-specific ligands areflanked by short glycine stretches to provide flexibility and embeddedinto a loop formed by two cystines. This is one of the classicalstrategies to incorporate ligands into a protein scaffold (Doi, N. andH. Yanagawa, 1998, Cell Mol. Life Sci., 54:394–404; Koivunen, E. et al.,1995, Biotechnology (NY), 13:265–70) and to guarantee their presentationat the protein surface. The biodistribution of the best variants istested in vivo in C57B1/6 mice based on Southern blots or PCR for vectorDNA in different organs. This mouse strain in known to be susceptible toinfection with P. berghei (Chatterjee, S. et al., 1995, Infect Immun.,63:4375–81).

K. Production of Adenovirus Vectors with Increased Tropism to TumorCells

A similar strategy is to insert two peptides obtained after selectionfor tumor tropism by displaying random peptides on filamentous phages.The first double cyclic peptide (RGD-4) proved to bind specifically tointegrins present on tumor vasculature (Ellerby H. M. et al., 1999, Nat.Med., 5:1032–8). The second peptide targets specific matrixmetalloproteinases associated with metastatic tumor cells as shown forthe breast cancer cell line MDA-MB-435 (Koivunen, E. et al., 1999, Nat.Biotechnol., 17:768–74). Tropism-modified vectors are tested in animalmodels with hepatic metastases derived from MDA-MB-435 cells (FIG. 28).

L. Development of a Peptide Display Technique Based on Adenoviruses

A synthetic peptide library is described that allows adenovirus vectorsto express random peptides in the G-H loop of the fiber knob domain. Thetechnique of a phage display library is optimized to generate a libraryof adenoviruses displaying random peptides in their fiber knob. Thislibrary of adenovirus variants is then screened for tropism to aparticular cell type in vitro and potentially in vivo. Theoligonucleotides encoding the peptide library employ a novel techniqueto assemble pre-synthesized trinucleotides representing the codons forall 20 amino acids. This will end the termination codons and assureoptimal codon usage and translation in human cells. To maintain arepresentative library size, a new “seamless” cloning strategy thatallows for insertion of the library into the wild-type Ad5 genomewithout introducing additional amino acids at the substitution site andwithout transformation into bacteria. Transfection into 293 cells isdone by conjugating the viral library DNA to carrier Ad5-basedadenovirus via polylysine to assure a 100% transfection efficiency.Importantly, the carrier adenoviral genome has its packaging signalflanked by lox sites preventing the packaging of carrier viral DNA afterinfection of 293 cells that express Cre recombinase (293/cre). Thelibrary is produced with E1-positive viruses depleted for CAR andintegrin tropism. Only variant that have successfully infected the celltype of interest will replicate, resulting in de novo produced virus.The sequence of the peptide ligand that conferred the particular tropismwill then be analyzed in do novo produced virus.

EXAMPLE III

Combination Novel Adenoviral Vector and Modified Fiber Protein

This example describes the following studies which combine thetechnology of the integrating adenovirus vector that is devoid of alladenoviral genes with the modified fiber protein that retargets thevector to quiescent HSC.

A. Transduction Studies with Re-Targeted Vectors in HSC:

In order to transduce quiescent HSC and integrate into chromosomal DNA,retargeted ΔAd.AAV^(fx) vectors are tested for reporter gene expression,and vector integration simultaneously while analyzing their clonogeniccapacity. The modified ΔAd.AAV^(fx) hybrid vectors contain genomesdevoid of all adenoviral genes (a “gutless” adenovirus vector) packagedinto Ad5 capsids with modified fibers. Rep may be incorporated intothese ΔAd.AAV^(fx) vectors to allow for site-specific integration intoAAVS1.

Transduction Studies:

Purified human CD34+ cells in IMDM/FCS+IL-3 and SCF are infected withdifferent doses of ΔAd.AAV^(fx)-BG (1–10⁷ genomes per cell). CD34+ cellsinfected with ΔAd.AAV^(fx)-βGal are cultured for 2 days in suspensionand sort β-Gal+ cells by FACS using FDG as substrate. This determinesthe infection efficiency. β-gal expressing cells are then submitted toclonogenic assays in semi-solid cultures (in two dishes per MOI) in thepresence of multiple cytokines. (IL-3, SCF, Epo, G-CSF, GM-CSF, IL-7,Tpo). A first set of semi-solid cultures can be evaluated after 7 days;another set can be analyzed after 14 days. Colonies that have formed insemisolid culture can be characterized by light microscopy andsubsequently stained with X-Gal staining. Most of the vector genomesshould remain episomal and can be lost with successive cell divisions.Thus, while most cells can be X-Gal positive at day 2 or day 7 afterinfection, most of the larger colonies (analyzed at day 14 p.i.) may notstain homogeneously for β-Gal. A representative number of X-Gal positiveand X-Gal negative colonies can be picked and analyzed for episomal andintegrated vector DNA. The outcome depends on the MOI used for infectionand the integration status of the vector. These studies determinewhether hybrid vectors can infect primitive progenitors.

Detailed Characterization of Hybrid Vector Integration:

CD34+ cells can be infected with ΔAd.AAV^(fx)-SNori (MOI 1–10⁷) andsubjected to G418 selection in methyl cellulose (MC) cultures in thepresence of growth factors (IL-3 and SCF). The resulting colonies are amixture of mainly myeloid cells. The number and morphology of G418resistant colonies can be determined after 2 weeks of selection. Thisstrategy may be disadvantageous in that the appropriate stem cell maynot divide and form G418 resistant colonies under the specific culturecondition used. Moreover, it may be difficult to perform G418 selectionon a population of heterogenous cells, which vary in their sensitivityto G418. Therefore, another set of ΔAd.AAV^(fx)-SNori infected CD34+cells can be cultured in methyl-cellulose (+IL-3, SCF) without G418selection. After 2–3 weeks, single colonies can be picked from both (w/and w/o G418) MC cultures, morphologically characterized, and analyzedfor integrated vector using the modified protocol developed forintegration studies in a small number of cells (see FIG. 8). Thisstrategy allows the assessment of whether hybrid vectors integrate intothe genome of CD34+ cells cultured in the presence of growth factors.This study gives us an idea about potential position effects affectingneo or {overscore (β)}gal expression from integrated vector copies andabout the structure of the integrated vector and the flankingchromosomal regions.

An Alternative Method to Confirm Vector Integration:

Fluorescence in situ hybridization (FISH) analysis, can be performed inindividual cells from MC colonies. CD34+ cells are cultured in MC in thepresence of growth factors to induce cell division and subsequentlytreated with colchicine. Metaphase chromosome spreads are analyzed withbiotin-ATP labeled probe specific for the {overscore (β)}Gal or SNorigene and a dioxigenin-UTP labeled probe for the human X-chromosome as aninternal control (provided by Christine Disteche, University ofWashington). Specific hybridization can be visualized with correspondinganti-biotin or anti-DIG antibodies labeled with different fluorochromes(e.g. FITC and Texas Red). Hybrid vector DNA may integrate asconcatemers, which would facilitate detection by FISH. This techniqueallows one to localize the chromosomal integration sites of hybridvectors.

Test Transduction into Quiescent Bone Marrow Subpopulations:

Hybrid vectors described so far can be tested to see whether quiescentCD34+ cells can be stably transduced. To avoid significant cellproliferation, purified CD34+ cells are cultured in serum free IMDMsupplemented with thrombopoietin (Tpo). Tpo can alone support thesurvival of stem cells without stimulating their active cellproliferation (Matsunaga, T. et al., 1998, Blood, 92:452–61;Papayannopoulou, T. et al., 1996, Experimental Hematology, 24:660–69).To analyze the proliferation status of CD34+ cells at the time point ofinfection with the hybrid vector ΔAd.AAV^(fx)BG, BrdU is added 2 hoursbefore infection to the culture medium. One set of cells are maintainedas suspension culture in IDAM containing Tpo only for two days. Anotherset of cells are grown in IDAM+Tpo supplemented with multiple cytokines.Forty eight hours after infection, CD34+ cells can be FACS sorted forbeta Gal expression using FDG. FDG positive cells can be furtheranalyzed for cellular DNA replication based on BrdU incorporation andfor specific CD34+subset markers. To do this, cytospins from FDG+ cellscan be submitted to immunofluorescence with BrdU specific antibodies andwith antibodies to specific cell surface markers (e.g. CD38, CD41).Alternatively, consecutive paraffin sections of the same cell can beanalyzed for (a) transgene expression by X-Gal staining, (b) DNAsynthesis based on BrdU incorporation, and (c) specific surface markers.This allows one to confirm that the culture conditions with Tpo aloneprevent significant genomic DNA replication and subsequent cellproliferation as well as to determine whether quiescent CD34+ cells canbe infected based on beta Gal expression in cells where BrdU labeling isabsent.

Test Hybrid Vectors Integration into Quiescent CD34+ Cells:

Two sets of CD34+ cells are infected. The first set of □Ad.AAV^(fx)SNoriinfected cells are cultured for 5–7 days in the presence of cytokines;the other set is cultured without cytokines. To maintain CD34+ cellviability without cytokines during this period, the cells are culturedin the presence of Tpo or underlaid with a stromal cell line (AFT024)(Moore, K. et al., 1997, Blood, 89:4337–47), which can maintain HSCviable for 4 to 7 weeks. After this specific time period, both sets aresubmitted to clonogenic assays (in the presence of multiple cytokines)either in combination with G418 selection or without selection. Singlecolonies are analyzed morphologically and submitted to genomic DNAanalysis (FIG. 8) to determine the vector integration status. Theultimate proof for stem cell transduction is the in vivosurvival/expansion assay. To do this, the CD34+ cells expressing betaGal are used for transplantation experiments, If the number of FDG+cells is not sufficient, total ΔAd.AAV^(fx)BG infected cells as well asall ΔAd.AAV^(fx)-SNori infected cells can be used directly withoutselection. Transplantation can be performed via tail vein injection intosublethally irradiated SCID NOD mice (Dao, M. A., et al., 1998, Blood,4, 1243–1255; Matsunaga, T. et al., 1998, Blood, 92:452–61). Atdifferent time points after transplantation (4 to 8 weeks), mice can besacrificed to obtain bone marrow cells which then can be cultured insuspension until various assays are performed for X-Gal and cell markersas described earlier. These cells also can be submitted to a secondarycolony assay in MC or secondary transplantation into SCID NOD mice.Furthermore, MC colonies derived from these cells can be analyzed forthe presence of integrated vector DNA by the method illustrated in FIG.8. The expression and integration data together allow conclusions aboutthe repopulation efficiency and about potential position effects.

B. Optimization of ΔAd.AAV^(fx) Vectors for γ r-globin Expression inHematopoietic Stem Cells:

One specific example of the invention is (a) to construct retargetedhybrid vector with the γ-globin as the transgene under the control oferythroid cell specific promoter, (b) to analyze the level and kineticsof γ-globin expression after transduction with hybrid vectors in invitro and in vivo assays, (c) if required, to protect gene expressionfrom position effects using γ-globin LCRs or insulators incorporatedinto hybrid vectors, and (d) to study whether γ-globin introns orheterologous introns can increase γ-globin expression.

Another central issue of the invention is to demonstrate that hybridvectors can accommodate larger transgenes than rAAV and retroviruses.The insert size limitation of these vectors is 5 kb. Transgene cassettesup to 8 kb can be inserted into hybrid vectors as described. The maximalinsert size may be about 14 kb, if hybrid vectors are produced on thebasis of E2a and/or E4 deleted rAd vectors in corresponding packagingcell lines. The maximal insert size in hybrid vectors is dictated by thepackaging limit of first generation vectors (Ad.AAV) (<36 kb) which arenecessary intermediates for hybrid virus production at large scale. Itis expected that stability and titer of Ad.AAV vectors with an 8 kbglobin gene cassette is comparable to the vector containing the 2.5–3.5kb cassette used in Ad.AAVBG, Ad.AAV1, and Ad.AAVSNori. The followingexample experiments address these issues.

Production of ΔAd.AAV^(fx) with Large Globin Expression Cassettes:

In order to improve the condition of sickle cell disease, the expressionlevel of the transferred γ-lobin gene must be at least 50% of that ofeach endogenous βgene. These levels of transgene expression can only beachieved by using optimal expression cassettes, including extended LCRsand intron containing gamma genes. (Forrester, W. C., et al., 1986,Proc. Natl. Acad. Sci. USA 83, 1359–1363; Fraser, P., et al., 1998,Curr. Opinion in Cell Bio., 10, 361–365; Grosveld, F., et a.l, 1998,Seminars in Hematology, 35, 105–111; Martin, D. et al., 1996, CurrentOpinion in Genetics and Development, 6:488–95), So far, most of theγ-globin expression cassettes are designed for retroviral and rAAVvectors, thus, less than 5 kb and have to be devoid of internal splicesites or poly adenylation signals. With integrating vectors describedherein, it is possible to go beyond this size limitation. This allowsone to improve γ-globin expression in bone marrow cells in terms of anadequate expression level and long term persistence. For this purpose,γ-globin constructs developed by Li et al (Emery, D. W., et al. 1999 HumGene Ther 10:877–88; Li, Q., et al. 1999. Blood 93:2208–16) or by Elliset al (Ellis, J., et al., 1996, EMBO J., 15, 562–568; Ellis, J., et al.,1997, Nucleic Acids Res. 25, 1296–1302) is chosen.

(i) The first cassette contains a γ-globin expression unit used inretroviral vectors. This allows for a direct comparison between the twosystems. This construct includes the beta promoter from −127 to the betainitiation codon, which is connected in frame with the gamma codingregion. This beta promoter is combined with the 300 bp HS40 derived fromthe human alpha globin locus, which acts as a strong enhancer for globinexpression. The globin gene is the 1.1 kb version with intron 1 andpartially deleted intron 2. A second cassette is generated containingthe HS40 beta promoter and gamma globin gene with the complete 3.3 kbgamma globin gene.

(ii) The second construct contains the 6.5 kb beta μLCR, which confer adominant chromatin opening activity and an adequate level of gammaglobin expression in transgenic mice. The LCR is linked to the short 1.1kb version of the gamma globin gene or the complete 3.3 kb gamma gene.

(iii) Additional globin expression cassette can be generated whichinclude insulators, MARs or SARs, as well as other elements that canimprove transgene expression from integrated vectors or in transgenicanimals, like introns derived from the HPRT or hGH genes (Chung, J. H.,et al., 1997, Proc. Natl. Acad. Sci. USA 94, 575–580; Dunaway, M., etal, 1993, Mol. Cell. Biol., 17, 182–189; Felsenfeld, G., et al., 1996,Proc. Natl. Acad. Sci. USA 93, 93840–9388; Klehr, D., et al., 1991,Biochemistry, 30, 1264–1270).

Transduction Studies with ΔAd.AAVfx-globin Vectors:

Transduction studies with globin-hybrid vectors are performed asdescribed earlier (Steinwaerder, D. S., et al. 1999. J Virol73:9303–13). Transduced CD34+ cells are submitted to differentiation incolony assays or analyzed in vivo expansion assays in SCID-NOD mice.MC-colonies or bone marrow cells from experimental mice are analyzed forglobin expression. Gamma-globin expression is measured using fluorescentanti-gamma-globin antibodies. RNAase protection studies can be performedto specifically quantitate gamma globin mRNA in comparison with -globinRNA. For these studies around 10⁴–10⁵ cells are needed per test.

Position Effects:

In the absence of the LCR, globin genes are subjected to strong positioneffects when they are transferred into cultured CD34+ cells orerythroleukemic lines (Fraser, P., et al., 1998, Curr. Opinion in CellBio., 10, 361–365; Grosveld, F., et a.l, 1998, Seminars in Hematology,35, 105–111). Another concern is that site-specific integration ofΔAd.AAV/rep vectors into AAVS1 may silence transgene expression. Ifsilencing happens, it can be overcome by incorporating LCRs such as the6.5 kb □ globin μLCR (Ellis, J., et al., 1996, EMBO J., 15, 562–568;Grosveld, F., et a.l, 1998, Seminars in Hematology, 35, 105–111) orinsulators into □Ad.AAV based expression units. Insulators are DNAelements that protect an integrated reporter gene from chromosomalposition effects or that block enhancer activated transcription from adownward promoter. Insulator elements are known for Drosophilamelanogaster genes (Gypsy, suppressor of Hairy wing, scs, scs′, Fab-7),for the chicken beta-globin gene (HS4) and for the T cell receptor(BEAD1; 14, 21.25). Specifically, the Drosophila gypsy or the betaglobin insulator can be inserted as two copies flanking the globinexpression cassette into hybrid vectors. The position effects can beexamined in transduced MC-colonies based on the analysis of integratedvector DNA (see FIG. 29) and gamma-globin mRNA quantification. Analogousstudies can be performed on transduced human bone marrow cells obtainedafter transplantation of infected CD34+ cells into SCID-NOD mice.

Intron Effects on Gamma-Globin Expression:

A number of reports reveal that the deletion of globin introns,particularly the second intron of the beta and gamma genes, decreaseglobin mRNA stability and thus the expression level (Antoniou, M. etal., 1998, Nucleic Acid Res., 26:721–9). RNA viruses such as onco-retro,lenti- and foami viruses are problematic as vehicles forintron-containing transgenes. Because ΔAd.AAV is a DNA virus, it shouldpackage globin introns and LCRs if necessary without the decreasedtiters and rearrangements observed with retroviral vectors.

APPENDIX I Human and Animal Adenoviruses Available from American TypeCulture Collection

-   1: Adenovirus Type 21 ATCC VR-1099-   2: SA18 (Simian adenovirus 18) ATCC VR-943-   3: SA17 (Simian adenovirus 17) ATCC VR-942-   4: Adenovirus Type 47 ATCC VR-1309-   5: Adenovirus Type 44 ATCC VR-1306-   6: Avian adenovirus Type 4 ATCC VR-829-   7: Avian adenovirus Type 5 ATCC VR-830-   8: Avian adenovirus Type 7 ATCC VR-832-   9: Avian adenovirus Type 8 ATCC VR-833-   10: Avian adenovirus Type 9 ATCC VR-834-   11: Avian adenovirus Type 10 ATCC VR-835-   12: Avian adenovirus Type 2 ATCC VR-827-   13: Adenovirus Type 45 ATCC VR-1307-   14: Adenovirus Type 38 ATCC VR-988-   15: Adenovirus Type 46 ATCC VR-1308-   16: Simian adenovirus ATCC VR-541-   17: SA7 (Simian adenovirus 16) ATCC VR-941-   18: Frog adenovirus (FAV-1) ATCC VR-896-   19: Adenovirus type 48 (candidate) ATCC VR-1406-   20: Adenovirus Type 42 ATCC VR-1304-   21: Adenovirus type 49 (candidate) ATCC VR-1407-   22: Adenovirus Type 43 ATCC VR-1305-   23: Avian adenovirus Type 6 ATCC VR-831-   24: Avian adenovirus Type 3 (Inclusion body hepatitis virus)-   25: Bovine adenovirus Type 3 ATCC VR-639-   26: Bovine adenovirus Type 6 ATCC VR-642-   27: Canine adenovirus ATCC VR-800-   28: Bovine adenovirus Type 5 ATCC VR-641-   29: Adenovirus Type 36 ATCC VR-913-   30: Ovine adenovirus type 5 ATCC VR-1343-   31: Adenovirus Type 29 ATCC VR-272-   32: Swine adenovirus ATCC VR-359-   33: Bovine adenovirus Type 4 ATCC VR-640-   34: Bovine adenovirus Type 8 ATCC VR-769-   35: Bovine adenovirus Type 7 ATCC VR-768-   36: Adeno-associated virus Type 2 (AAV-2H) ATCC VR-680-   37: Adenovirus Type 4 ATCC VR-4-   38: Adeno-associated virus Type 3 (AAV-3H) ATCC VR-681-   39: Peromyscus adenovirus ATCC VR-528-   40: Adenovirus Type 15 ATCC VR-661-   41: Adenovirus Type 20 ATCC VR-662-   42: Chimpanzee adenovirus ATCC VR-593-   43: Adenovirus Type 31 ATCC VR-357-   44: Adenovirus Type 25 ATCC VR-223-   45: Chimpanzee adenovirus ATCC VR-592-   46: Chimpanzee adenovirus ATCC VR-591-   47: Adenovirus Type 26 ATCC VR-224-   48: Adenovirus Type 19 ATCC VR-254-   49: Adenovirus Type 23 ATCC VR-258-   50: Adenovirus Type 28 ATCC VR-226-   51: Adenovirus Type 6 ATCC VR-6-   52: Adenovirus Type 2 Antiserum: ATCC VR-1079-   53: Adenovirus Type 6 ATCC VR-1083-   54: Ovine adenovirus type 6 ATCC VR-1340-   55: Adenovirus Type 3 ATCC VR-847-   56: Adenovirus Type 7 ATCC VR-7-   57: Adenovirus Type 39 ATCC VR-932-   58: Adenovirus Type 3 ATCC VR-3-   59: Bovine adenovirus Type 1 ATCC VR-313-   60: Adenovirus Type 14 ATCC VR-15-   61: Adenovirus Type 1 ATCC VR-1078-   62: Adenovirus Type 21 ATCC VR-256-   63: Adenovirus Type 18 ATCC VR-1095-   64: Baboon adenovirus ATCC VR-275-   65: Adenovirus Type 10 ATCC VR-11-   66: Adenovirus Type 33 ATCC VR-626-   67: Adenovirus Type 34 ATCC VR-716-   68: Adenovirus Type 15 ATCC VR-16-   69: Adenovirus Type 22 ATCC VR-257-   70: Adenovirus Type 24 ATCC VR-259-   71: Adenovirus Type 17 ATCC VR-1094-   72: Adenovirus Type 4 ATCC VR-1081-   73: Adenovirus Type 16 ATCC VR-17-   74: Adenovirus Type 17 ATCC VR-18-   75: Adenovirus Type 16 ATCC VR-1093-   76: Bovine adenovirus Type 20 ATCC VR-314-   77: SV-30 ATCC VR-203-   78: Adenovirus Type 32 ATCC VR-625-   79: Adenovirus Type 20 ATCC VR-255-   80: Adenovirus Type 13 ATCC VR-14-   81: Adenovirus Type 14 ATCC VR-1091-   82: Adenovirus Type 18 ATCC VR-19-   83: SV-39 ATCC VR-353-   84: Adenovirus Type 11 ATCC VR-849-   85: Duck adenovirus (Egg drop syndrome) ATCC VR-921-   86: Adenovirus Type 1 ATCC VR-1-   87: Chimpanzee adenovirus ATCC VR-594-   88: Adenovirus Type 15 ATCC VR-1092-   89: Adenovirus Type 13 ATCC VR-1090-   90: Adenovirus Type 8 ATCC VR-1368-   91: SV-31 ATCC VR-204-   92: Adenovirus Type 9 ATCC VR-1086-   93: Mouse adenovirus ATCC VR-550-   94: Adenovirus Type 9 ATCC VR-10-   95: Adenovirus Type 41 ATCC VR-930-   96: CL ATCC VR-20-   97: Adenovirus Type 40 ATCC VR-931-   98: Adenovirus Type 37 ATCC VR-929-   99: Marble spleen disease virus (Hemorrhagic enteritis virus)-   100: Adenovirus Type 35 ATCC VR-718-   101: SV-32 (M3) ATCC VR-205-   102: Adenovirus Type 28 ATCC VR-1106-   103: Adenovirus Type 10 ATCC VR-1087-   104: Adenovirus Type 20 ATCC VR-1097-   105: Adenovirus Type 21 ATCC VR-1098-   106: Adenovirus Type 25 ATCC VR-1103-   107: Adenovirus Type 26 ATCC VR-1104-   108: Adenovirus Type 31 ATCC VR-1109-   109: Adenovirus Type 19 ATCC VR-1096-   110: SV-36 ATCC VR-208-   111: SV-38 ATCC VR-355-   112: SV-25 (M8) ATCC VR-201-   113: SV-15 (M4) ATCC VR-197-   114: Adenovirus Type 22 ATCC VR-1100-   115: SV-23 (M2) ATCC VR-200-   116: Adenovirus Type 11 ATCC VR-12-   117: Adenovirus Type 24 ATCC VR-1102-   118: Avian adenovirus Type 1 (Chicken-Embryo Lethal Orphan)-   119: SV-11 (M5) ATCC VR-196-   120: Adenovirus Type 5 ATCC VR-5-   121: Adenovirus Type 23 ATCC VR-1101-   122: SV-27 (M9) ATCC VR-202-   123: Avian adenovirus Type 2 (GAL) ATCC VR-280-   124: SV-1 (M1) ATCC VR-195-   125: SV-17 (M6) ATCC VR-198-   126: Adenovirus Type 29 ATCC VR-1107-   127: Adenovirus Type 2 ATCC VR-846-   128: SV-34 ATCC VR-207-   129: SV-20 (M7) ATCC VR-199-   130: SV-37 ATCC VR-209-   131: SV-33 (MIO) ATCC VR-206-   132: Avian adeno-associated virus ATCC VR-865-   133: Adeno-associated (satellite) virus Type 4 ATCC VR-646-   134: Adenovirus Type 30 ATCC VR-273-   135: Adeno-associated (satellite) virus Type 1 ATCC VR-645-   136: Infectious canine Hepatitis (Rubarth's disease)-   137: Adenovirus Type 27 ATCC VR-1105-   138: Adenovirus Type 12 ATCC VR-863-   139: Adeno-associated virus Type 2 (molecularly cloned)-   140: Adenovirus Type 7a ATCC VR-848

1. A recombinant, double-stranded, adenovirus vector, wherein the vectorcomprises: a. an adenovirus left inverted terminal repeat sequence; b.an adenovirus packaging sequence; c. a first adenoviral-associatedinverted terminal repeat sequence; d. a first inverted repeat sequence;e. a heterologous promoter sequence which mediates transcription in adirection towards the adenoviral left inverted terminal repeat sequencein part a; f. a foreign gene sequence; g. a second inverted repeatsequence; h. a second adenoviral-associated inverted terminal repeatsequence; i. a gene sequence that mediates replication of an adenovirusin a transduced cell; and j. an adenovirus right inverted terminalrepeat sequence; wherein the adenovirus packaging sequence is located onthe one strand of the double-stranded vector; and wherein the other ofthe two strands of the double-stranded vector comprises a nucleotidesequence encoding a modified adenoviral fiber protein which alters thetropism of the adenovirus vector, and wherein the modified adenoviralfiber protein is a modified fiber knob, a modified fiber tail or amodified fiber shaft.
 2. A recombinant, double-stranded, adenovirusvector wherein the vector comprises: a. an adenovirus left invertedterminal repeat sequence; b. an adenovirus packaging sequence; c. afirst adenoviral-associated inverted terminal repeat sequence; d. afirst inverted repeat sequence; e. a heterologous promoter sequencewhich mediates transcription in a direction away from the adenoviralleft inverted terminal repeat sequence in part a; f. a foreign genesequence; g. a second inverted repeat sequence; h. a secondadenoviral-associated inverted terminal repeat sequence; i. a genesequence that mediates replication of an adenovirus in a transducedcell; and j. an adenovirus right inverted terminal repeat sequence;wherein the adenovirus packaging sequence is located on one strand ofthe double-stranded vector of the vector; and wherein the other of thetwo strands of the double-stranded vector comprises a nucleotidesequence encoding a modified adenoviral fiber protein which alters thetropism of the adenovirus vector, and wherein the modified adenoviralfiber protein is a modified fiber knob, a modified fiber tail or amodified fiber shaft.
 3. The adenoviral vector of claim 1 or 2, whereinthe modified fiber knob, the modified fiber tail or the modified fibershaft is from an adenoviral serotype that differs from the serotype ofthe left or right adenoviral inverted terminal repeat sequence.
 4. Theadenoviral vector of claim 1 or 2, wherein the modified fiber knob, themodified fiber tail or the modified fiber shaft is from adenoviralserotypes Ad3, Ad7, Ad9, Ad11 or Ad35.
 5. The adenoviral vector of claim1 or 2, wherein the modified fiber knob binds a cell surface protein ona target cell of interest.
 6. The adenoviral vector of claim 1 or 2,wherein the modified fiber knob is modified in the G-H loop region orH-I loop region.
 7. The adenoviral vector of claim 1 or 2, wherein themodified fiber knob comprises a heterologous peptide ligand whichreplaces the G-H loop region or H-I loop region.
 8. The adenoviralvector of claim 7, wherein the heterologous peptide ligand is an RI orRII protein from malaria circumsporozoite surface protein (CS).
 9. Theadenoviral vector of claim 8, wherein the RI protein from malariacircumsporozoite surface protein (CS) comprises the amino acid sequenceKLKQPG (SEQ ID NO.:12).
 10. The adenoviral vector of claim 8, whereinthe RII protein from malaria circumsporozoite surface protein (CS)comprises the amino acid sequence EWSPCSVTCGNGIQVRIK (SEQ ID NO.:13).11. The adenoviral vector of claim 7, wherein the heterologous peptideligand comprises the amino acid sequence: a. LGGKPDQ (SEQ ID NO.:15); b.LNGCGSC (SEQ ID NO.:16); c. LNGCGSGC (SEQ ID NO.:17); or d.LNGCGXXXXXXXXXXGC (SEQ ID NO.:18).
 12. The adenoviral vector of claim 1or 2 which infects hepatocytes, bone marrow cells, stem cells or breastcancer cells.
 13. The adenoviral vector of claim 1 or 2, wherein themodified fiber shaft has a shortened length.
 14. The adenoviral vectorof claim 1 or 2, wherein the adenoviral packaging sequence and the leftand right adenoviral inverted terminal repeat sequences are from thesame adenoviral serotype.
 15. The adenoviral vector of claim 1 or 2,wherein the adenoviral packaging sequence and the left and rightadenoviral inverted repeat sequences are from serotype Ad5.
 16. Theadenoviral vector of claim 1 or 2, wherein the foreign gene sequenceencodes a therapeutic gene product, a selectable gene product, or areporter gene product.
 17. The adenoviral vector of claim 1 or 2,wherein the therapeutic gene product is gamma globin or human alpha-1antitrypsin.
 18. The adenoviral vector of claim 1 or 2, wherein theselectable gene product is neomycin, ampicillin, penicillin,tetracycline or gentamycin.
 19. The adenoviral vector of claim 1 or 2,wherein the reporter gene product is green fluorescent protein, betagalactosidase or alkaline phosphatase.
 20. The adenoviral vector ofclaim 1 or 2, further comprising an insulator element sequence.
 21. Theadenoviral vector of claim 1 or 2, further comprising a bacterial originof replication.
 22. The adenoviral vector of claim 1 or 2, furthercomprising a nucleotide sequence encoding a rep78 protein.
 23. Theadenoviral vector of claim 1 or 2, wherein the gene sequence thatmediates replication of an adenovirus in the transduced cell is selectedfrom a group consisting of E2 and E4; E1, E2 and E4; and E2, E3 and E4.24. The adenoviral vector of claim 1 or 2, wherein the foreign genesequence comprises a 5′ portion of the foreign gene sequence.
 25. Theadenoviral vector of claim 1 or 2, wherein the foreign gene sequencecomprises a 3′ portion of the foreign gene sequence.